Induction of apoptic or cytotoxic gene expression by adenoviral mediated gene codelivery

ABSTRACT

The present invention generally relates to viral vectors and their use as expression vectors for transforming human cells, both in vitro and in vivo. More particularly, the present invention relates to adenoviral vectors containing propapoptotic genes and their use in cancer therapy.

BACKGROUND OF THE INVENTION

This application claims priority to and specifically incorporates byreference, the content of U.S. Provisional Application Ser. No.60/077,541 filed Mar. 11, 1998. The entire text of each of theabove-referenced disclosures is specifically incorporated by referenceherein without disclaimer. The government owns rights in the presentinvention pursuant to grant number CA70907 from the National Institutesof Health.

1. Field of the Invention

The present invention relates generally to viral vectors and their useas expression vectors for transforming human cells, both in vitro and invivo. More specifically, the invention relates to adenoviral expressionconstructs comprising a proapoptotic member of the Bcl-2 gene family.

2. Description of Related Art

Adenoviral vectors have become one of the leading vectors for genetransfer, particularly in gene therapy contexts. These vectors have beenstudied rigorously in both in vitro and in vivo contexts because of theability to generate high titer stocks, their high transductionefficiency and their ability to infect a variety of tissue types indifferent species. In addition, the availability of cell lines tocomplement defects in adenoviral replication functions provides for theuse of replication defective mutants carrying, in the place of selectedstructural genes, recombinant inserts of interest.

Several studies have demonstrated the ability of adenovirus-mediatedwild-type p53 replacement gene therapy to induce a G₁ cell cycle arrestand/or apoptosis in malignant cells carrying p53 gene mutations. Thoughthe mechanism of G₁ arrest via p21 and the cyclin-dependent kinasepathway has been widely studied, little is known of the mechanisms bywhich wild-type p53 induces apoptosis. It appears that p53 inducesapoptosis, at least in part, by up-regulating proapoptotic members ofthe Bcl-2 family of proteins.

The Bcl-2 family of proteins and ICE-like proteases have beendemonstrated to be important regulators and effectors of apoptosis inother systems. Apoptosis, or programmed cell death, is an essentialoccurring process for normal embryonic development, maintaininghomeostasis in adult tissues, and suppressing carcinogenesis (Kerr etal., 1972). The Bcl-2 protein, discovered in association with follicularlymphoma, plays a prominent role in controlling apoptosis and enhancingcell survival in response to diverse apoptotic stimuli (Bakhshi et al.,1985; Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al.,1985; Tsujimoto and Croce, 1986). The evolutionarily conserved Bcl-2protein now is recognized to be a member of a family of related proteinswhich can be categorized as death agonists or death antagonists.

Subsequent to its discovery, it was shown that Bcl-2 acts to suppresscell death triggered by a variety of stimuli which will be discussed indetail. Also, it now is apparent that there is a family of Bcl-2 celldeath regulatory proteins which share in common structural and sequencehomologies. These different family members have been shown to eitherpossess similar functions to Bcl-2 or counteract Bcl-2 function andpromote cell death.

One such family member having Bcl-2 counteracting function is Bax. Bax,Bcl-2 associated X protein, is a death agonist member of the Bcl-2family of proteins (Oltvai et al., 1993). It has been suggested that Baxmay function as a primary response gene in a p53 regulated apoptoticpathway (Miyashita et al., 1994). Indeed, it has been shown that thereis a p53 consensus binding region in the promoter region of theproapoptotic Bax gene (1995). Bax mRNA and protein expression areincreased following induction of p53. However, the observed induction ofp53-dependent apoptosis in Bax knock out mice clearly indicates thatother pathways or proteins are involved. Bak, a Bcl-2 homologue, isexpressed in a variety of tissues and has been demonstrated to induceprogram cell death independent of Bax expression (Krajewski et al.,1996; Chittenden et al., 1995). The accumulation of Bak protein in cellsinfected with Adp53, may be an additional mechanism by which p53 caninduce programmed cell death.

However, a recent report has demonstrated an increase in Bcl-x_(L)expression following wild-type p53 expression in the human colorectalcancer cell line HT29 (Merchant et al., 1996). The authors hypothesizethat this increase expression may lead to an inhibition of program celldeath pathways and accounted for lack of p53-induced apoptosis observedin these cells. Another potential problem with p53 therapy is that theamount of viral material administered provides risks of host celltoxicity and/or immune response. Thus, any method which would increasethe effect of p53 at low doses, or circumvent the need for high viraldoses, would be advantageous.

Given that p53 gene therapy is a powerful tool in the fight againstcancer, therapeutic compositions that may augment or complement p53 willserve to improve the currently available cancer therapy regimens.Indeed, compositions that provide the apoptotic effect of p53 withoutthe need for p53 itself would be additionally useful.

SUMMARY OF THE INVENTION

The present invention generally is related the use of viral vectorscontaining propapoptotic genes and their use in cancer therapy, in orderto induce an apoptotic effect in cancer cells to either augment,complement or bypass the need for p53 based therapy.

In order to achieve the objectives of the present invention, aparticular embodiment provides an adenoviral expression constructcomprising a nucleic acid encoding a proapoptotic member of the Bcl-2gene family and a first promoter functional in eukaryotic cells whereinthe nucleic acid is under transcriptional control of the first promoter.In particularly preferred embodiments, the proapoptotic Bcl-2 gene is aBax, Bak, Bim, Bik, Bid or Bad gene. In certain embodiments, it iscontemplated that the adenoviral expression construct may furthercomprise a second nucleic acid encoding a second gene. In particularinstances the second nucleic acid is under the control of the firstpromoter.

In particularly preferred embodiments, the proapoptotic Bcl-2 gene andthe second nucleic acid are separated by an IRES. In alternativeembodiments, the second nucleic acid is under the control of a secondpromoter operative in eukaryotic cells. It is contemplated that thepromoter employed herein may be any promoter used in the production ofexpression constructs. In particularly preferred embodiments thepromoter may be selected from the group consisting of CMV IE, SV40 IE,RSV, β-actin, tetracycline regulatable and ecdysone regulatable.

In certain defined aspects, the second gene may encode a proteinselected from the group consisting of a tumor suppressor, a cytokine, areceptor, inducer of apoptosis, and differentiating agents. By“differentiating agents,” the present application refers to the functionof bcl-2 family members in the induction of differentiation in cells.Thus, the cells are not induced to die via apoptosis, but terminallydifferentiate and stop growing, which is equally effective as a cancertreatment. In particularly preferred embodiments, the tumor suppressormay be selected from the group consisting of p53, p16, p21, MMAC1, p73,zac1, C-CAM, BRCAI and Rb. In certain embodiments, the inducer ofapoptosis is selected from the group consisting of Harakiri, Ad E1B andan ICE-CED3 protease. In those embodiments employing a cytokine, thecytokine may be selected from the group consisting of IL-2, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14,IL-15, TNF, GMCSF, β-interferon and γ-interferon. In those embodimentswhere the second gene is a receptor, the receptor may be selected fromthe group consisting of CFTR, EGFR, VEGFR, IL-2 receptor and theestrogen receptor. It is contemplated that the second nucleic acid maybe an antiapoptotic member of the Bcl-2 gene family or an oncogene, thesecond nucleic acid being positioned in an antisense orientation withrespect to the promoter. In more preferred embodiments, theantiapoptotic member of the Bcl-2 gene family is Bcl-2 or Bcl-x_(L). Inembodiments in which the second gene is an oncogene, the oncogene may beselected from the group consisting of ras, myc, neu, raf erb, src, fins,jun, trk, ret, gsp, hst, and abl.

In defined embodiments, the expression construct is areplication-deficient adenovirus. In preferred aspects, the adenoviruslacks at least a portion of the E1 region. In other embodiments, theadenovirus further lacks the E3 coding region. In preferred embodiments,the expression construct further comprises a polyadenylation signal. Inparticular embodiments, the nucleic acid may be a cDNA, or genomic DNA.

In particularly preferred embodiments, the proapoptotic member of theBcl-2 family is Bax. In other preferred embodiments, the proapoptoticmember of the Bcl-2 family is Bak. In more preferred embodiments, theBax gene expresses a truncated Bax protein. In more preferredembodiments, the truncated Bax protein comprises an intact death domain.In other preferred embodiments, the truncated Bax protein comprises SEQID NO:2. In other preferred embodiments, the truncated Bax proteincomprises a BH3 region.

Also contemplated by the present invention is a pharmaceuticalcomposition comprising a first adenoviral expression constructcomprising a promoter functional in eukaryotic cells and a first nucleicacid encoding a proapoptotic member of the Bcl-2 gene family, whereinthe first nucleic acid is under transcriptional control of the promoterand a pharmaceutically acceptable buffer, solvent or diluent.

In particularly preferred embodiments, the proapoptotic Bcl-2 familygene is a Bax, Bak, Bik, Bid, or Bad gene. In other preferredembodiments, the promoter may be selected from the group consisting ofCMV IE, SV40 IE, RSV, β-actin, tetracycline regulatable and ecdysoneregulatable. In other embodiments, the pharmaceutical composition mayfurther comprise a second expression construct encoding a second nucleicacid encoding a second gene operatively linked to a second promoter. Incertain aspects, the expression construct encoding the proapoptotic genefurther comprises a second nucleic acid encoding a second gene. Thesecond nucleic acid may be under the control of the first promoter. Inalternative embodiments, the second nucleic acid is under the control ofa second promoter operative in eukaryotic cells. The second gene mayencode a protein selected from the group consisting of a tumorsuppressor, a cytokine, a receptor, inducer of apoptosis, anddifferentiating agents. In particularly preferred embodiments, thesecond nucleic acid is an antiapoptotic member of the Bcl-2 gene familyor an oncogene, the second nucleic acid being positioned in an antisenseorientation with respect to the promoter.

In preferred embodiments, the present invention further contemplates amethod for treating a subject with cancer comprising the steps ofproviding an adenoviral expression construct comprising a nucleic acidencoding a proapoptotic member of the Bcl-2 gene family and a firstpromoter functional in eukaryotic cells wherein the nucleic acid isunder transcriptional control of the first promoter; and contacting theexpression construct with cancer cells of the subject in a manner thatallows the uptake of the expression construct by the cells, whereinexpression of the proapoptotic gene results in the treatment of thecancer. By “treatment,” the present invention refers to any event thatdecreases the growth, kills or otherwise abrogates the presence ofcancer cells in a subject. Such a treatment may also occur by inhibitionof the metastatic potential or inhibition of tumorigenicity of the cellso as to achieve a therapeutic outcome.

In other preferred aspects, the method further comprises contacting thecancer cell with a further cancer therapeutic agent. In particularlypreferred embodiments, the cancer therapeutic agent may be selected fromthe group consisting of tumor irradiation, chemotherapeutic agent, asecond nucleic acid encoding a cancer therapeutic gene. In definedembodiments, the chemotherapeutic agent is a DNA damaging agent selectedfrom the group consisting of verapamil, podophyllotoxin, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, taxol, transplatinum, 5fluorouracil, vincristin,vinblastin and methotrexate. In alternative embodiments, the radiationis selected from the group consisting of X-ray radiation, UV-radiation,γ-radiation, or microwave radiation. In other defined embodiments, thecancer therapeutic agent comprises a second nucleic acid. The secondnucleic acid may be a cDNA or genomic DNA.

In particular embodiments of the present invention, the secondexpression construct is selected from the group consisting of anadenovirus, an adeno-associated virus, a vaccinia virus and aherpesvirus. In other embodiments, the contacting is effected byregional delivery of the expression construct. In alternativeembodiments, the contacting is effected by local delivery of theexpression construct. In still further embodiments, the contacting maybe effected by direct injection of a tumor with the expressionconstruct. In particularly preferred embodiments, the contactingcomprises delivering the expression construct endoscopically,intratracheally, intralesionally, percutaneously, intravenously,subcutaneously or intratumorally to said subject. In certainembodiments, the method may further comprise the step of tumorresection. The tumor resection may occur prior to or after thecontacting. The tumor resection may be performed one, two, three or moretimes. In particularly preferred embodiments, the cancer being treatedmay be selected from the group consisting of lung, breast, melanoma,colon, renal, testicular, ovarian, lung, prostate, hepatic, germ cancer,epithelial, prostate, head and neck, pancreatic cancer, glioblastoma,astrocytoma, oligodendroglioma, ependymomas, neurofibrosarcoma,meningia, liver, spleen, lymph node, small intestine, blood cells,colon, stomach, thyroid, endometrium, prostate, skin, esophagus, bonemarrow and blood.

The present invention also provides a method of inhibiting the growth ofa cell comprising the steps of providing an adenoviral expressionconstruct comprising a nucleic acid encoding a proapoptotic member ofthe Bcl-2 gene family and promoter functional in eukaryotic cellswherein the nucleic acid is under transcriptional control of the firstpromoter; and contacting the expression construct with the cell in anamount effective to inhibit the growth of the cell wherein expression ofthe proapoptotic gene by the cell results in a decrease in the growth ofthe cell relative to the growth of the cell in the absence of theproapoptotic gene.

In preferred embodiments, the cell is a cancer cell. In other preferredembodiments, the inhibition of growth comprises killing of the cancercell. In other embodiments, the inhibition of growth comprises aninhibition of metastatic growth of the cancer cell. In definedembodiments, the cancer cell may be selected from the group consistingof lung, breast, melanoma, colon, renal, testicular, ovarian, lung,prostate, hepatic, germ cancer, epithelial, prostate, head and neck,pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma,ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node,small intestine, blood cells, colon, stomach, thyroid, endometrium,prostate, skin, esophagus, bone marrow and blood. In other embodiments,the cell is located within a mammal.

The present invention also provides a method of inducing apoptosis in acell comprising the steps of providing an adenoviral expressionconstruct comprising a nucleic acid encoding a proapoptotic member ofthe Bcl-2 gene family and promoter functional in eukaryotic cellswherein the nucleic acid is under transcriptional control of the firstpromoter; and contacting the expression construct with the cell in anamount effective to kill the cell; wherein expression of theproapoptotic gene by the results in an increase in the rate of death ofsaid cell relative to the growth of said cell in the absence of saidproapoptotic gene. In particularly preferred embodiments, theproapoptotic member of the Bcl-2 gene family is a Bax, Bak, Bim, Bik,Bid or Bad gene. In more preferred embodiments, the proapoptotic memberof the Bcl-2 gene family is a truncated Bax gene. In other preferredembodiments, the proapoptotic member of the Bcl-2 gene family is atruncated Bak gene.

Also contemplated by the present invention is a nucleic acid encoding atruncated Bax gene. In particular embodiments, the Bax gene comprises anucleic acid sequence of SEQ ID NO:1. In other embodiments, the Bax geneencodes a protein having an amino acid sequence of SEQ ID NO:2. Inparticularly preferred aspects the truncated Bax gene encodes a proteincomprising a BH3 region. In alternative preferred embodiments, thetruncated Bax gene encodes a protein comprising an intact death domain.

In yet another embodiment, the present invention further contemplates anadenoviral expression construct comprising a nucleic acid encoding atruncated Bax gene and a first promoter functional in eukaryotic cellswherein the nucleic acid is under transcriptional control of the firstpromoter. The adenoviral expression construct may further comprise asecond nucleic acid encoding a second gene. The second gene may be underthe control of the first promoter. In alternative embodiments, thesecond gene may be under the transcriptional control of a secondpromoter. In a further alternative, the truncated Bax gene and thesecond nucleic acid may be separated by an IRES.

In yet another embodiment, the present invention further contemplates anadenoviral expression construct comprising a nucleic acid encoding a bakgene and a first promoter functional in eukaryotic cells wherein thenucleic acid is under transcriptional control of the first promoter. Theadenoviral expression construct may further comprise a second nucleicacid encoding a second gene. The second gene may be under the control ofthe first promoter. In alternative embodiments, the second gene may beunder the transcriptional control of a second promoter. In a furtheralternative, the truncated Bak gene and the second nucleic acid may beseparated by an IRES.

In other embodiments there is provided, a method for expressing apolypeptide in a target cell comprising introducing into the target cella first vector comprising a coding region for a polypeptide under thecontrol of a first promoter inducible by an inducer polypeptide notexpressed in the target cell and a second vector comprising a codingregion for the inducer polypeptide under the control of a secondpromoter active in the target cell. In certain embodiments, the firstand second vectors are viral vectors. In other embodiments, the firstand said second vectors are non-viral vectors. In yet other embodiments,the first vector is a viral vector and the second vector is a non-viralvector, or the first vector is a non-viral vector and the second vectoris a viral vector. It is contemplated that the second promoter is aconstitutive promoter, an inducible promoter or a tissue specificpromoter.

In certain embodiments, the viral vectors are the same or different andmay be selected from the group consisting of an adenoviral vector, aherpesviral vector, a retroviral vector, an adeno-associated viralvector, a vaccinia viral vector or a polyoma viral vector.

It is contemplated in one embodiment that the first vector and thesecond vector are introduced into the target cell at a ratio of 1:1,respectively. In other embodiments, the first vector and the secondvector are introduced into the target cell at a ratio of 2:1,respectively. In still other embodiments, the first vector is introducedat 900 MOI and the second vector at 1500 MOI into the target cell.

In another embodiment, the first promoter is GAL4 and the inducerpolypeptide is GAL4/VP16, respectively. It is contemplated in otherembodiments, that the first promoter can be selected from the groupconsisting of the ecdysone-responsive promoter, and Tet-On™ and theinducer ecdysone or muristeron A and doxycycline, respectively.

In particular embodiments, the target cell is a hyperproliferative cell,a pre-malignant cell or a malignant cell. In embodiments where thetarget cell is malignant, it is contemplated that the malignant cell maybe selected form the group consisting of a lung cancer cell, a prostatecancer cell, a brain cancer cell, a liver cancer cell, a breast cancercell, a skin cancer cell, an ovarian cancer cell, a testicular cancercell, a stomach cancer cell, a pancreatic cancer cell, a colon cancercell, an esophageal cancer cell, head and neck cancer cell.

In certain embodiments, the first and second vectors are introduced intothe target cell at the same time. In one embodiment, the first vector isintroduced into the target cell prior to the second vector. In otherembodiments, the second vector is introduced into the target cell within24 hours, within 12 hours, within 6 hours, within 3 hours or within 1hour of the first vector. In another embodiment, the second vector isintroduced into the target cell prior to the first vector. It iscontemplated, that the first vector is introduced into the target cellwithin 24 hours, within 12 hours, within 6 hours, within 3 hours orwithin 1 hour of the second vector.

In other embodiments, the target cell is further contacted with a DNAdamaging agent. It is contemplated that the DNA damaging agent may beradiotherapy or chemotherapy.

In one embodiment, the second promoter is an inducible promoter and theinducing factor is present in the target cell. In another embodiment,the second promoter is an inducible promoter and the inducing factor isadded to the target cell. In particular embodiments, it is contemplatedthat one or both of the vectors further comprise a polyadenylationsignal.

In certain embodiments, the polypeptide expressed in the target cell iscytotoxic. It is contemplated that the cytotoxic polypeptide mayselected from the group consisting of an inducer of apoptosis, acytokine, a toxin, a single chain antibody, a protease and an antigen.It is further contemplated that the inducer of apoptosis may be selectedfrom the group consisting of Bax, Bak, Bik, Bim, Bid, Bad and Harakiri.In preferred embodiments, the inducer of apoptosis is Bax. In otherembodiments, it is contemplated that the cytokine may be selected formthe group consisting of oncostatin M, TGF-β, TNF-α and TNF-β. In yetother embodiments, the toxin may be selected form the group consistingof ricin A-chain, diphtheria toxin A-chain, pertussis toxin A subunit,E. coli enterotoxin A subunit, cholera toxin A subunit and pseudomonastoxin c-terminal. In particularly preferred embodiments, the toxin isdiphtheria toxin A-chain.

In one embodiment, a kit comprising a first vector comprising a firstpromoter, inducible by an inducer polypeptide, a multipurpose cloningsite 3′ to the first promoter in a suitable container and a secondvector comprising a coding region for the inducer polypeptide under thecontrol of a second promoter active in the target cell in suitablecontainer. In another embodiment, the first vector further comprises aregion coding for a polypeptide under control of the first promoter. Inyet another embodiment, the second promoter is an inducible promoter andthe kit further comprises an agent that induces the second promoter in asuitable container means.

Also contemplated is a method of treating a disease comprisingintroducing into cells of a subject having a disease a first vectorcomprising a coding region for the therapeutic polypeptide under thecontrol of a first promoter inducible by an inducer polypeptide notexpressed in the target cell and a second vector comprising a codingregion for the inducer polypeptide under the control of a secondpromoter active in the target cell. In one embodiment, the disease maybe selected from the group consisting of lung cancer, prostate cancer,brain cancer, liver cancer, breast cancer, skin cancer, ovarian cancer,testicular cancer, stomach cancer, pancreatic cancer, colon cancer,esophageal cancer and head and neck cancer. In another embodiment, thetherapeutic polypeptide may be selected from the group consisting ofBax, Bak, Bik, Bim, Bid, Bad, Harakiri, ricin A-chain, diphtheria toxinA-chain, pertussis toxin A subunit, E. coli enterotoxin A subunit,cholera toxin A subunit, pseudomonas toxin c-terminal, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSFoncostatin M, TGF-β, TNF-α, TNF-β and G-CSF.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1 Schematic depiction of the protein structures of the Bcl-2 familymembers. BH1, BH2, BH3, and BH4 are the conserved homology domains. TMindicates the transmembrane domain, NH2 indicated the amino terminaldomain, and the PEST domain represents the region which is correlated toan early response gene product and is associated with rapid proteinturnover. GRS is grouped with the anti-apoptotic family members,however, its role in apoptosis is not currently known.

FIG. 2A and FIG. 2B. Western blot analysis of CPP32 and Parp expression.Control non-infected cells and cells following infection withAd5/CMB/p53 were collected and subjected to western blot analysis usingmonoclonal antibody against CPP32 (FIG. 2B) or polyclonal antibodyagainst parp (FIG. 2A). Fifty micrograms of protein was analyzed bySDS-PAGE and visualized by western blotting using the ECLchemiluminescence system. Image shown is an optical scan of arepresentative film exposure from one of three studies. The arrowsindicate expected location of CPP32 and Parp cleavage product.

FIG. 3A and FIG. 3B. Effect of Ad5/CMV/p53 gene transfer on cell cycleregulation and induction of apoptosis. Cell cycle analysis and TUNELwere performed on cells which were treated with control vector DL312 orPBS or infected with Ad5/CMV/p53 and collected at 6 h intervalsfollowing infection. Cells were tripsinized at the reported time pointfixed and analyzed for DNA content by perpidium iodine staining andanalyzed for TUNEL labeling by fluorescence using flow cytometry.Infection with Ad5/CMV/p53 resulted in a increase in G₁ population ofcells and an increase in the 2N population of cells (FIG. 3A).Additionally infection with Ad5/CMV/p53 resulted in an increasedpopulation of TUNEL-labeled cells consistent with increases in apoptoticdeath (FIG. 3B).

FIG. 4A, FIG. 4B, and FIG. 4C. FACS analysis to measure apoptosis inMCF-7 cells (FIG. 4A), SKBr3 cells (FIG. 4B) and MDA-MD-468 cells (FIG.4C). Cells were either uninfected, infected with an empty adenoviralvector control, an adenovirus vector containing the truncated bax gene.

FIG. 5. Plasmid map of the Supercos vector.

FIG. 6. Plasmid map of pCOS/LJ07.

FIG. 7. Plasmid map of pCOS/Ad/LJ17.

FIG. 8. Plasmid map of pCMV/Bak.

FIG. 9. Plasmid map of pCOS/Ad-Bak.

FIG. 10. Schematic of cloning adenovirus genome into cosmid.

FIG. 11. Schematic of construction of recombinant adenovirus in E. coli.

FIG. 12. Schematic of production of recombinant adenovirus.

FIG. 13. Schematic of adenovirus-mediated gene co-transfer. Theexpression cassettes for the transgene (bax) and the transactivator(GAL4/VP16) are cloned into separate vectors. The expression of thetransgene is then induced after co-infecting a target cell with the twovectors.

FIG. 14. Apoptosis profiles after induction of bax gene expression.Nuclear fragmentation detected by staining with Hoechst 33432. Thetreatment for each sample is indicated above each panel.

FIG. 15. In vivo induction of bax gene expression. Nuclear fragmentationdetected by hematoxylin and eosin staining of liver sections from micetreated with (a) PBS, (b) Ad/GT-Bax+Ad/CMV-GFP, (c)Ad/GT-Bax+Ad/PGK-GV16, and (d) Ad/GT-LacZ+Ad/CMV-GV16.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Cancer accounts the death of over half a million people each year in theUnited States alone. The causes for cancer are multifactorial, however,it is known that aberrations in controlled cell death result inuncontrolled cell proliferation and hence contribute to many cancer.

The p53 gene is well-recognized as possessing tumor suppressorcapabilities and mutations in wild-type p53 are correlated to a varietyof cancers. However, the interaction of p53 with other cellular factorsis not well characterized, in fact, many of these factors remainundefined. It is not surprising that, in light of the lack ofsignificant information on p53 function, there is an incompleteunderstanding of the pathways through which p53 regulates tumordevelopment. Nevertheless, p53-based gene therapy has been remarkablyeffective in inducing cell cycle arrest and/or apoptosis in malignantcells carrying p53 gene mutations.

There now is a great deal of evidence that the apoptotic effect of p53is mediated through the members of the proapoptotic Bcl-2 family. It hasbeen shown that the p53 dependent expression of Bax is induced inslow-growing apoptotic tumors. Further, tumor growth appearsaccelerated, and apoptosis is decreased, in Bax-deficient mice. Thissuggests that Bax is required for a full p53-mediated response (Yin etal., 1997). The present invention, for the first time, provides evidencethat proapoptotic Bcl-2 genes in adenoviral vectors can be used todecrease, diminish, inhibit or otherwise abrogate the growth of cancercells.

The present invention employs, in one embodiment, an adenoviralexpression construct comprising a gene that encodes a truncated Baxprotein. As discussed herein below, the Bcl-2 family of proteinsconsists of death antagonists and death agonists that regulate apoptosisand compete through dimerization. All members of the Bcl-2 family ofproteins contain one or more Bcl-2 homology domains (BH). It appearsthat there are at least 4 BH domains, referred to as BH1, BH2, BH3 andBH4. The competition between the proapoptotic and antiapoptotic membersis mediated at least in part, by competitive dimerization betweenselective pairs of antagonists and agonist molecules. Mutagenesisstudies revealed that intact BH1 and BH2 domains of antagonists arerequired for repression of cell death. Conversely, the BH3 domain of Baxis the domain responsible for conferring the death agonist activity toBcl proteins. Thus, in preferred embodiments, the present invention usesa truncated Bax protein having an intact “death domain.” Of course otherBcl proteins such as Bak, Bid, Bik, that comprise the death domain willalso be useful in the adenoviral constructs of the present invention.

In the present invention, the overexpression of the proapoptoticmediator Bax has been demonstrated in cancer cell lines transduced withan adenoviral Bax construct. Morphologically, apoptosis was seen within4 days post-transduction. Thus, the present invention demonstrates thatBax induces apoptosis in cancer cell lines and provides evidence thatadenoviral constructs containing Bax and/or other proapoptotic Bcl-2gene family members will be useful components of a cancer therapyregimen. Methods of producing and using such compositions are discussedin further detail below.

In another embodiment, an adenoviral-mediated gene co-transfer system isdescribed, that permits the regulated expression of cytotoxic geneproducts for use in treating hyperproliferative disease. In oneembodiment, a first vector carrying a gene encoding a toxic product isunder the control of a promoter, not active in the target source. Asecond vector, comprises a transactivator gene, wherein thetransactivator protein product activates transcription from the promoterin the first expression vector. The choice of promoter on the secondexpression vector can be selected for use on an as needed basis (e.g.,tissue specificity). It is contemplated further, that the co-transfersystem can be used with any expression vector or combination thereof(e.g., viral, plasmid, plasmid shuttle vector, cosmid), introduced viaany method of gene transfer desired (i.e., viral or non-viral) and usedfor both in vivo and in vitro.

A. The Bcl-2 Gene Family and Apoptosis

Apoptosis is an essential process required for normal embryonicdevelopment, maintenance of adult tissue homeostasis and the suppressionof carcinogenesis. Apoptosis has been defined as a type of cell deathwhich complements mitosis in the regulation of cell populations (Kerr etal., 1972). Apoptosis can occur as a result of both physiologic andpathologic conditions and is believed to be, in many developmentalcontexts, a programmed event. The sequence of events begins with nuclearand cytoplasmic condensation and ends with the release and phagocytosisof apoptotic bodies (Kerr et al., 1972).

A major advance in understanding the regulation of apoptosis came withthe discovery of the Bcl-2 proto-oncogene from the t(14;18) chromosomaltranslocation breakpoint in follicular lymphoma (Bakhshi et al., 1985;Cleary and Sklar, 1985; Cleary et al., 1986; Tsujimoto et al., 1985;Tsujimoto and Croce, 1986). Bcl-2 acts to suppress cell death triggeredby a variety of stimuli and, it is now apparent that there is a familyof Bcl-2 cell death regulatory proteins which share in common structuraland sequence homologies. These different family members have been shownto either possess similar functions to Bcl-2 or counteract Bcl-2function and promote cell death. These cell death regulators arediscussed in further detail herein below.

In mammalian development, Bcl-2 and Bcl-2 family members have been shownto play a role in morphogenesis and normal development. During murinefetal development Bcl-2 is expressed in tissues derived from all threegerm layers; however, as the fetus matures, Bcl-2 expression becomesrestricted (Novack and Korsmeyer, 1994). Similar observations were seenin human fetal tissues in that Bcl-2 was expressed in a wide variety oftissue types and expression became restricted as the fetus matured(LeBrun et al., 1993; Chandler et al., 1994). Bcl-2 was detected in thehuman fetal thymus, hematopoietic cells, endocrine glands, andhormonally regulated tissues and differential expression of Bcl-2 familymembers occurs during neuronal differentiation. Bcl-x_(L) and Bcl-2 areboth expressed in neurons of the developing human fetus, however,Bcl-x_(L) expression persists throughout fetal development and intoadulthood whereas Bcl-2 expression diminishes between wk 20-39 ofgestation (Yachnis et al., 1997).

Although Bcl-2 protein is widely expressed in embryonic tissues (Novackand Korsmeyer, 1994; Lu et al., 1993), absence of Bcl-2 protein in Bcl-2null mice does not interfere with normal prenatal development (Veis etal., 1993). However, postnatally, these mice display growth retardation,smaller ears, and polycystic kidneys, and most die within several monthsdue to kidney failure. In the Bcl-2 deficient mice, which eventuallybecome ill, the thymus and spleen are atrophic due to massive lymphocyteapoptosis. Also, Bcl-2 null thymocytes are more susceptible to undergoapoptosis following γ-irradiation or treatment with dexamethasone(Kamada et al., 1995; Nakayama et al., 1994).

The tissue distribution of Bcl-2 expression also suggests that Bcl-2plays a role in survival in various cell types (Hockenbery et al.,1991). Immunohistochemistry reveals that Bcl-2 is expressed in cellsthat regenerate such as the stem cells or in cells that are long lived.In the lymphatic system, Bcl-2 is strongly expressed in the thymicmedulla where the T-cells which have survived negative and positiveselection reside, and in the areas of lymph nodes associated withmaintenance of plasma cells and memory B-cells (Hockenbery et al., 1991;Nunez et al., 1991). In non-hematopoietic tissues, Bcl-2 is restrictedto cells that undergo self renewal such as the basal layer of the skin,the crypt cells of the small and large intestine, and in long livedcells such as the neurons. Bcl-2 also is expressed in tissues such asbreast duct epithelium and prostate epithelium which undergohyperproliferation or involution at the influence of the hormone orgrowth factors (Hockenbery et al., 1991; McDonnell et al., 1992).

The Bcl-2 family continues to expand with the discovery of new members.Table 1 summarizes the Bcl-2 family of cell death regulators. TABLE 1Human Bcl-2 Family Members Genbank Family Gene mRNA Amino Acid ProteinSize Chromosome Sequence Member Size (Kb) Size (Kb) Residues (kD)Localization Function Identifiers Bcl-2 230 6.5 239 25 18q21Anti-Apoptotic M14745 Bcl-x_(L) ND 2.7 233 31 ND Anti-Apoptotic Bcl-w 22 3.7 193 22 ND Anti-Apoptotic Mcl-1 ND 3.8 350 37.3 1q21Anti-Apoptotic Al ND 1.4* 172 20** ND Anti-Apoptotic Bfl-1 ND 0.6* 17520*** ND Anti-Apoptotic Bax α  4.5 1.0 192 21 19q13.3-13.4 Pro-ApoptoticL22473 Bax β 1.5 218 24 ND L22474 Bax γ  41****  4.5** ND L22475 Bax δ1.5 143**** ? ND U19599 ND Bcl-x_(s) ND 1.0 170 19 ND Pro-ApoptoticZ23116; L20122 Bak 1  6 2.4 211 23  6 Pro-Apoptotic U16811; U23765 Bak 220 U16812; U23765 Bak3 11 Bad ND 1.1* 204 22 ND Pro-Apoptotic AF031523Bid ND 1.1* 195 23 ND Pro-Apoptotic U75506 Bik ND 1.0 160 18 NDPro-Apoptotic U34584 GRS ND 0.8 175 ? 15q24-25 ND Harakiri ND 0.7  9116***** ND Pro-Apoptotic U76376ND, not determined.*cDNA;**predicted size of protein;***size of Ha-Tagged protein;****predicted amino acid length;*****size of the Flagged protein.The known Genbank sequences are listed and are specifically incorporatedherein by reference in order to provide additional disclosure of thesequences of the genes of the Bcl family.

It is commonly accepted that tumorigenesis is a multistep process whichmay involve chromosomal abnormalities and the deregulated expression ofproto-oncogenes (Bishop, 1991). This is particularly evident inhematolymphoid neoplasms where chromosomal translocations may result inthe activation of a proto-oncogene. Translocations involve the breakageand reunion of chromosomes where part of one chromosome breaks off andbecomes reattached to another chromosome. Such translocations aredescribed by a notation that indicates which two chromosomes have beenrecombined. For example, t(9:22) indicates that a translocation hasoccurred between chromosome 9 and chromosome 22. Further delineation ofthe exact regions or genes that are involved in the translocation leadto the identification of the resulting gene fusions or proto-oncogenesinvolved in each particular translocation event. Certain chromosomaltranslocations are associated with activation of oncogenes that lie nearthe breakpoint of the chromosome.

Characterization of the t(9;22) and t(8;14) translocations in chronicmyelogenous leukemia (Nowell and Hungerford, 1960; Rowley, 1973) andBurkitt's lymphoma (Manalov and Manolova 1972; Zech et al., 1976),respectively, provided a paradigm for the deregulation ofproto-oncogenes during multistep carcinogenesis.

B. Bcl-2 Family Members

Many Bcl-2 family member proteins have now been identified (FIG. 1).These Bcl-2 homologues can be broadly categorized as death antagonistsand death agonists. The growing list of Bcl-2 gene family members allshare highly conserved domains referred to as Bcl-2 homology domain 1and 2 (BH1 and BH2) (Oltvai et al., 1993; Yin et al., 1994; Yin et al.,1995) or domains B and C, respectively (Hanada et al., 1995; Tanaka etal., 1993). These homology domains seem to be important for Bcl-2 toform heterodimeric complexes with the family members and to carry outits anti-apoptotic function (Yin et al., 1994; Hanada et al., 1995; Yanget al., 1995a; Korsmeyer et al., 1993; Sedlak et al., 1995). Forexample, mutations in BH1 and BH2 prevent Bcl-2 from formingheterodimeric complexes with the Bcl-2 homologue Bax and can abrogatethe survival function of Bcl-2 (Yin et al., 1994). The Bcl-2 protein canalso form homodimers with itself via its NH2 terminal region called theBH4 domain which spans residues 11 through 33 (Hanada et al., 1995).

Thus, as stated earlier, the Bcl-2 family members are divided intoproapoptotic and antiapoptotic genes. The proapoptotic genes includeBax, Bak, Bcl-x_(S), Bad, Bik, Bid and Harakiri. The antiapoptotic genesinclude Bcl-2, Bcl-x_(L), Mcl-1, A1, Bcl-w and GRS (FIG. 1). Each ofthese genes are discussed in further detail herein below.

i. Bcl-2

The t(9;22) results in the formation of a bcr-abl fusion gene andchimeric protein (Shrivelman et al., 1985) while the t(8;14) results inthe inappropriate expression of c-myc (Dalla-Favera et al., 1982; Taubet al., 1984; Nishikura et al., 1983). Both of these molecular eventsresult in augmented cellular proliferation (Langdon et al., 1986).

Bcl-2 was discovered as a novel transcriptional element by itsassociation with the t(14;18) reciprocal chromosomal translocationcommonly found in follicular lymphoma (Bakhshi et al., 1985; Cleary andSklar, 1985, Tsujimoto et al., 1984). Bcl-2 was shown to be a uniqueoncogene in that its deregulation did not result in an increase in cellproliferation, but rather enhancement of cell survival (Vaux et al.,1988; Hockenbery et al., 1990; McDonnell et al., 1989). Thus, Bcl-2represents a class of oncogene that enables neoplastic growth bysuppressing cell death (McDonnell, 1993a).

The Bcl-2 gene, is comprised of three exons and spans approximately 230Kb. The open reading frame is in exon 2 and 3, and encodes a 25 kDintegral membrane protein (Seto et al., 1988; Zutter, et al., 1991). Themessage can be alternatively spliced to give two transcripts, Bcl-2α andthe truncated Bcl-2α that lacks the C-terminus region (Tsujimoto andCroce, 1986). Bcl-2 possesses a very hydrophobic stretch of 23 aminoacids at the C-terminus which serve as a transmembrane domain(Hockenbery et al., 1990). Bcl-2 protein localizes to the nucleus, roughER, and mitochondria. In mitochondria, the protein is localized to thecontact zone of the inner and outer membranes of the mitochondrialmembrane where the transport of materials from the cytosol into themitochondrial matrix occurs (Hockenbery et al., 1990; deJong et al.,1992).

Bcl-2 is normally expressed in pro and mature B-cells, but isdownregulated in pre and immature B lymphocytes (Merino et al., 1994).This differential expression points to the survival role of Bcl-2 in Blymphocyte development. High levels of Bcl-2 are needed to ensure thesurvival of pro-B-cells and mature B-cells in order to maintain apopulation of functional lymphocytes. But low levels of Bcl-2 arenecessary for cells, which do not express functional surface Ig or areself reactive, to undergo apoptosis. Also in T-cells, Bcl-2 is expressedat low levels in double positive thymocytes undergoing negative andpositive selection, and at high levels in mature single positive T-cellswhich have survived the selection (Gratiot-Deans et al., 1993). Thus,Bcl-2 seems to have an important role in lymphocyte development(McDonnell et al., 1989; McDonnell et al., 1990; McDonnell andKorsmeyer, 1991;).

The Bcl-2-Ig transgenic mouse model demonstrates that deregulation ofBcl-2 gene causes initially a polyclonal expansion of mature B-cellswhich can progress to an aggressive monoclonal malignancy with anacquisition of additional gene deregulation, thus confirming themultistep nature of carcinogenesis (McDonnell, 1993b). In humans also,follicular lymphoma can progress to a high grade lymphoma following theacquisition of t(8; 14) translocations and c-myc gene deregulation,albeit this appears to be an uncommon event (Gawerky et al., 1988).

It also has been demonstrated that Bcl-2 plays a role in the suppressionof p53-mediated cell death. Splenic mononuclear cells obtained fromBcl-2-Ig mice, which possess wild-type p53, displayed rates of apoptosiscomparable to cells obtained from p53 knockout mice followingγ-irradiation (Marin et al., 1994). Together, these results and theresults of others utilizing transformed cell lines indicate that Bcl-2is capable of blocking p53 mediated cell death induction (Marin et al.,1994; Wang et al., 1993; Chiou et al., 1994).

Mutations in the conserved domains of p53 were uncommon in the lymphomasarising in the Bcl-2-Ig transgenic mice suggesting that there is noselective advantage of acquiring p53 mutations when Bcl-2 isoverexpressed (Marin et al., 1994). Additionally, the Bcl-2-Igtransgenic and p53 knockout murine models were further utilized todetermine the extent of genetic complementation between p53 and Bcl-2.In p53 KO/Bcl-2 hybrid mice, tumor latency and incidence were unchangedwhen compared to individual parental strains of mice (Marin et al.,1994). Many human tumors, such as breast and prostate, also demonstratethat there is an inverse correlation between the presence of p53mutations and Bcl-2 expression (Silvstrini et al., 1994; McDonnell etal., 1997).

ii. Bax

Bax (SEQ ID NO:3=cDNA; SEQ ID NO:4=wild-type protein), “Bcl-2 associatedX protein”, is a death agonist member of the Bcl-2 family of proteins.Discovered by co-immunoprecipitation with Bcl-2, it was the first Bcl-2homologue to be identified (Oltvai et al., 1993). The 4.5 Kb Bax genemaps to 19q13.3-13.4 and is comprised of six exons (Apte et al., 1995).It shares 21% identity and 43% similarity with Bcl-2. The most conservedregions between the two molecules are within the BH1 and BH2 domainsencoded by exons 4 and 5, respectively (Oltvai et al., 1993).

Multiple forms of Bax protein can result from various splicingalternatives. The most prevalent from is Bax-α, whose 1.0 Kb RNA encodesa 192 amino acid, 21 kD transmembrane protein. The 24 kD cytosolic Bax-βlacks the transmembrane segment and is encoded by 1.5 Kb RNA transcript.A third form, Bax-γ lacks the exon 2 and can undergo alternativesplicing of intron S to yield 1.0 and 1.5 Kb RNA transcripts (Olsen etal., 1996). Yet another alternatively spliced form of Bax, Baxδ, has theC-terminal transmembrane anchor as well as the BH1 and BH2 domains (Apteet al., 1995). The functional role of these Bax variants remains to beelucidated.

The Bax gene promoter contains four p53 binding sites and the expressionof Bax is upregulated at the transcriptional level by p53 (Miyashita andReed, 1995). A temperature sensitive p53 mutant transfected into amyeloid cell line was associated with increased Bax mRNA after shiftingto the permissive temperature (Zhan et al., 1994). Also in cellsobtained from p53-null mice, the level of Bax proteins was found to belower (Miyashita et al., 1994). Moreover, following apoptosis inductionby ionizing radiation, the Bax mRNA was upregulated only in the cellline that possesses wild-type p53 (Zhan et al., 1994). These datasuggest that Bax may function as a primary response gene in a p53regulated apoptotic pathway (Miyashita et al., 1994). However,thymocytes from the Bax knockout mice were not diminished in theircapacity to undergo apoptosis after γ-irradiation, a pathway driven byp53 (Knudson et al., 1995). Bax expression can also be modulated byother factors. The mRNA level has been shown to be downregulated inmyeloid leukemia cell lines treated with IL-6 and/or dexamethasone(Lotem and Sachs, 1995). The half life of Bax mRNA can be increased incell lines expressing higher levels of Bcl-2 (Miyashita et al., 1995).However, this increase in stability of Bax mRNA by Bcl-2 protein appearsto be tissue specific.

Mutational analysis has shown that the BH1 and BH2 domains of Bax arenot required for heterodimerization with Bcl-2, nor is the NH₂ terminalamino acids needed for Bax homodimerization, unlike the homodimerizationrequirement for Bcl-2. Rather a stretch of amino acids spanning residues59-101 in the BH3 domain was shown to be essential in both thehomodimerization and heterodimer complex formation with Bcl-2 (Zha etal., 1996a). Additionally, in contrast to Bcl-2, Bax can function in itsmonomeric form to accelerate cell death (Simonian et al., 1996). Bax canheterodimerize with other Bcl-2 related proteins, including Bcl-x_(L),Mcl-1, and A1 (Sedlak et al., 1995). The “rheostat” model has beenproposed to explain the role of Bcl-2 family member interactions incontrolling cell death. This model suggests that the relative amounts ofBcl-2 and Bax may determine the susceptibility of a cell to undergoapoptosis (Korsmeyer et al., 1993). According to this model, when Bcl-2is in excess, Bcl-2/Bax heterodimers predominate and cell death isinhibited. Conversely, when Bax is in excess, Bax homodimers predominateand the cell becomes susceptible to cell death induction followingexposure to an apoptotic stimulus.

The tissue distribution of Bax protein is more widespread than Bcl-2.(Krajewski et al., 1994a). The immunohistochemical staining of murinetissues has revealed that the expression of Bcl-2 and Bax overlap insome tissues, and that Bax is not always expressed at high levels incompartments marked by a high turnover rate. For example, Bax, as wellas Bcl-2, are expressed in the thymic medulla but not in the thymiccortex, despite high numbers of cortical thymocytes which undergoapoptosis. Also, a high level of Bax protein is observed in neurons,cells that have a long life. However, in certain tissues such as colonicepithelium, gastric glands, and secretory epithelial cells of prostate,Bax expression corresponds to the cells that are susceptible toundergoing apoptotic cell death (Krajewski et al., 1994a).

Evidence that apoptosis is not absolutely dependent on the expression ofBax is also apparent from an analysis of the Bax knockout mice. In thesemice the absence of Bax is associated with either tissue specifichyperplasia or hypoplasia (Knudson et al., 1995). For example, there wasan increase in number of resting mature B-cells and thymocytes causinghyperplasia in the spleen and thymus. However, the male Bax knockoutmice were infertile due to atrophic testes, resulting from theabrogation of spermatogenesis (Knudson et al., 1995).

Recent evidence suggests that Bax may play a role as a tumor suppressor.Normally Bax-α is expressed at high levels in breast tissue but is notdetectable, or expressed at low levels, in breast cancers (Bargou etal., 1995). Furthermore, in metastatic breast cancer, patients withreduced Bax expression showed poor response to chemotherapy (Krajewskiet al., 1995a). Transgenic mice have been generated, which express atruncated form of the SV40 T antigen (Tgl21) resulting in inactivationof the retinoblastoma protein but not p53. Tgl21 mice bearing targeteddisruptions of either the p53 gene or the Bax gene exhibited anincreased rate of brain tumor formation compared to Tgl21 mice withintact p53 or Bax genes (Yin et al., 1997). Also frequent frame shiftmutations of Bax were found in microsatellite mutator phenotype (MMP)colon adenocarcinomas, suggesting that the wild-type Bax gene may play atumor suppressor role in colorectal carcinogenesis (Rampino et al.,1997).

iii. Bcl-x

Bcl-x was initially isolated from chicken lymphoid cells using a murineBcl-2 cDNA probe under low stringency conditions (Boise et al., 1993).The Bcl-x gene shares 44% identity with Bcl-2. Bcl-x was shown tointeract with other members of the Bcl-2 family in a manner similar tothat shown for Bcl-2 when analyzed by the yeast two-hybrid system (Satoet al., 1994). Two human Bcl-x cDNAs have been cloned (Boise et al.,1993). Bcl-x_(L) (long form) is a 31 kD protein, with an open readingframe of 233 amino acid. This form of Bcl-x contains the BH1 and BH2domains. The Bcl-x_(L) cDNA was found to be co-linear with the genomicsequence denoting the absence of mRNA splicing. Bcl-x_(s) (short form)encodes a 170 amino acid, 19 kD protein. The carboxy-terminal 63 aminoacids encoding the BH1 and BH2 domains are deleted from a 5′ splice sitewithin exon 1 of the Bcl-x gene (Boise et al., 1993). A thirdalternative splice variant of Bcl-x has been isolated from a murine cDNAlibrary, Bcl-x_(β), (Gonzalez-Garcia et al., 1994). Bcl-x_(β), encodes a209 amino acid protein that results from an unspliced first coding exonand lacks the carboxy-terminal 19 hydrophobic amino acids necessary fortransmembrane insertion.

Both the level and pattern of expression of Bcl-x differ from that ofBcl-2. The levels of Bcl-x expression are generally higher than Bcl-2 inall tissues examined except for the lymph nodes where Bcl-2 ispredominant (Krajewski et al., 1994a). Bcl-x_(L) is mainly expressed inthe cells of the central nervous system, kidney, and bone marrow(Gonzalez-Garcia et al., 1994, Rouayrenc et al., 1995). Both Bcl-x_(L)and Bcl-x_(s), but not Bcl-2 are expressed in CD34⁺, CD38⁻, lin⁻hematopoietic precursors (Park et al., 1995). However, the subcellulardistribution of Bcl-x protein is similar to Bcl-2 in that it localizesto mitochondria and the nuclear envelope. This suggests that thefunction of the two proteins may be similar (alez-Garcia et al., 1994,).

Further insight into the role of Bcl-x during development was obtainedfrom Bcl-x deficient mice (Motoyama et al., 1995). Heterozygous micedeveloped normally while homozygous, knockout mutants die atapproximately day 13 of gestation. The Bcl-x knockout embryos displayextensive apoptosis involving post-mitotic neurons of the developingbrain, spinal cord, dorsal root ganglia, and hematopoietic cells in theliver. Additionally, lymphocytes from Bcl-x deficient mice showeddiminished maturation. The life span of immature lymphocytes but notmature lymphocytes was shortened. This data indicates that Bcl-x isrequired for the embryonic development of the nervous and hematopoieticsystems.

Similar to Bcl-2, Bcl-x_(L) was shown to confer resistance to apoptosisinduction following growth factor deprivation. However, Bcl-x_(s)counteracted the ability of Bcl-2 to block apoptosis (Boise et al.,1993). Although Bcl-x_(L) and Bcl-2 initially seemed to have the samefunctions, several observations suggest that biologically these twoproteins are not completely overlapping. The tissue distribution ofBcl-2 and Bcl-x are not identical and the phenotypes' of thecorresponding knockout strains of mice are substantially different.Furthermore, it has been shown that WEHI-231 cells can be protected fromapoptosis induced by surface IgM cross-linking by enforced Bcl-x_(L)expression while enforced Bcl-2 expression exerts no such protectiveeffect (Choi and Boise, 1995; Gottschalk et al., 1994).

The crystalline structure of Bcl-x has expanded the inventors' insightinto the potential mechanisms of function of Bcl-2 family members(Muchmore et al., 1996). Bcl-x structure was shown to consist of twocentral hydrophobic α helices surrounded by two amphipathic helices(Muchmore et al., 1996). Interestingly, the conserved BH1, BH2 and BH3domains were in spatial proximity and formed a hydrophobic cleft. Thiscleft is believed to form a binding site for other Bcl-2 family members(Muchmore et al., 1996). Evidence in favor of this hypothesis wasprovided when Bcl-x and a 16 residue bak peptide derived from the BH3domain were co-crystallized. The heterodimeric crystal structurerevealed that the bak BH3 domain interacts with the hydrophobic cleftmade by the BH1, BH2, and BH3 domains of Bcl-x (Sattler et al., 1997).The crystal structure of Bcl-x was also found to resemble thetranslocation domain of the diphtheria toxin and colicins (Muchmore etal., 1996). This similarity in structure implies similarity in functionand indicates that Bcl-2 family members can be considered channelforming proteins capable of regulating the transmembrane trafficking ofmolecules involved in signaling cell death.

iv. Bak

Bak (Bcl-2-homologous antagonist/killer) was first cloned from humanheart and Epstein-Barr transformed human B-cell cDNA libraries(Chittenden et al., 1995; Kiefer et al., 1995; Farrow et al., 1995).There are three closely related bak genes (bak-1, 2, and 3) which arelocated on chromosome 6 (bak-1), chromosome 20 (bak-2) and chromosome 11(bak-3). The bak genes contain at least three exons and span 6 Kb. Bakis a 211 amino-acid, 23 kD protein which shares 53% amino-acid identitywith Bcl-2. It possesses the same hydrophobic carboxy-terminal domain asBcl-2 and Bcl-x_(L), which suggests that bak is an integral membraneprotein. In contrast to Bcl-2, bak is expressed at high levels in thekidney, pancreas, liver, and fetal heart, as well as adult brain (Kieferet al., 1995). Similar to Bax in the intestine, bak expression isstrongest in the cells in the luminal surface where most apoptosis isoccurring. However, in a colorectal carcinoma cell line, only bakexpression was shown to be modulated following apoptosis induction,indicating that bak may play a primary role in enterocyte apoptosis(Moss et al., 1996). This contention is further supported by theobservation that bak expression is reduced in colorectal adenocarcinomasamples. Therefore, a downregulation of bak may facilitate theaccumulation of neoplastic cells in the early stages of colorectaltumorigenesis (Krajewski et al., 1996). Bak was shown to accelerate celldeath following IL-3 withdrawal (Chittenden et al., 1995; Kiefer et al.,1995), but inhibits apoptosis induced by serum withdrawal and menadionetreatment (Chittenden et al., 1995).

v. Bad

Bad (Bcl-x_(L)/Bcl-2 associated death promoter homologue) a novel memberof the Bcl-2 family that was identified as a Bcl-2 interacting proteinusing the yeast two hybrid system (Yang et al., 1995b). The full-lengthBad cDNA sequence encodes a novel 204 amino acid protein with apredicted molecular weight of 22 kD. Bad shares only limited homologywith known Bcl-2 family members in the BH1 and BH2 domains. However, thefunctionally significant W/YGR triplet in BH1, the W at position 183,the WD/E at the exon junction in BH2 and the spacing between BH1 and BH2domains is conserved. Unlike many other Bcl-2 family members, Bad doesnot contain a transmembrane anchor domain.

Bad was shown to heterodimerize with Bcl-2 and Bcl-x in vivo usingco-immunoprecepitation. Bad's interaction with either Bcl-2 or Bcl-x candisplace Bax from the heterodimers. Significantly, this was shown toreverse the death repressor activity of Bcl-x, but not of Bcl-2.However, Bad does not appear to interact with Bax, Mcl-1, or A1 nor,apparently, does Bad form homodimers (Yang et al., 1995b). Recentstudies have shown that Bad may function in intracellular signaltransduction pathways. Upon IL-3 stimulation of an IL-3 dependenthematopoietic cell line, Bad becomes rapidly phosphorylated at twoserine residues and is prevented from forming heterodimeric complexeswith Bcl-x_(L). The phosphorylated Bad is found to be complexed with14-3-3, a phosphoserine binding protein which regulates protein kinases,and is sequestered in cytosol (Zha et al., 1996b). Therefore, only thenon-phosphorylated Bad is heterodimerized with the membrane boundBcl-x_(L) and counters the anti-apoptotic activity of Bcl-x_(L). One ofthe models to explain the apoptotic activity of Bad is that in itsnon-phosphorylated form, Bad binds to membrane associated Bcl-x_(L)which releases Bax to enhance cell death (Zha et al., 1996b). Anotherlink between the phosphorylation event and the apoptotic pathway wasshown when it was found that in vitro, Bad is phosphorylated bymitochondrial membrane targeted Raf-1, but not by the plasma membranetargeted Raf-1. Moreover, Bcl-2 was shown to target Raf-I tomitochondrial membrane which resulted in phosphorylation of Bad and thesubsequent enhancement of cell survival (Wang et al., 1996a).

vi. Mcl-1

Mcl-1 (human myeloid cell differentiation protein) was identified bydifferentially screening cDNA library of the human myeloid leukemia cellline, ML-1, following induction by phorbol 12-myristate 13-acetate (TPA)(Kozopas et al., 1993). Mcl-1 has also been detected in normalperipheral blood B cells after treatment with IL4 and anti-IgM. Mcl-1 isan early response gene, that reduces its expression immediatelyfollowing differentiation induction (Kozopas et al., 1993; Yang et al.,1995). A study done using a yeast two-hybrid assay indicates that Mcl-1interacts strongly and selectively with Bax, but not with any otherBcl-2 family members (Sedlak et al., 1995; Sato et al., 1994).

Mcl-1 shares sequence homology to Bcl-2 in the BH1 and BH2 domains andhas a carboxy-terminal transmembrane anchor domain (Yang et al., 1995).In addition, the Mcl-1 protein possesses PEST sequences (Kozopas et al.,1993), which correlate with the its role as an early response geneproduct (Yang et al., 1995). The human Mcl-1 gene maps to chromosome 1band q21 (Craig et al., 1994), an area often involved in chromosomalabnormalities in neoplastic and preneoplastic diseases (Atkin, 1986;Gendler et al., 1990; Testa, 1990).

Mcl-1 protects against apoptosis induced by constitutive expression ofc-myc or Bax (Reynolds et al., 107). However, in the 5AHSmyc cell line,Mcl-1 overexpression is not as effective as Bcl-2 overexpression inpreventing myc-mediated cell death (Reynolds et al., 1994). It has beenproposed that Mcl-1 may function as an alternative to Bcl-2 insituations where Bcl-2 cannot block apoptosis or in tissues lackingBcl-2 expression. For example, in normal peripheral blood B cellstreated with agents which promote survival (IL-4, anti-μ, and TPA) orenhance rates of cell death (TGFβ1 and forskolin), upregulation of Mcl-1correlates with cell survival and downregulation of Mcl-1 precedes celldeath. In contrast, levels of Bcl-2 expression are not modulated underthe same experimental conditions (Lomo et al., 1996).

Additionally, the tissue distribution of Mcl-1 and Bcl-2 expression showsignificant differences such as brain and spinal cord neurons in whichBcl-2 predominates compared to skeletal muscle, cardiac muscle,cartilage and liver where Mcl-1 predominates over Bcl-2 (Krajewski etal., 1995b). Similarly, Mcl-1 levels in normal lymph nodes are highestin germinal centers, where the rate of apoptosis is high. In contrast,Bcl-2 is most intense in the mantle zone. It has been postulated thatMcl-1 temporarily blocks cell death until suppression such as Bcl-2 areupregulated (Krajewski et al., 1994b).

vii. A1

A1 was identified by differentially screening a cDNA library of normalperipheral blood B cells and after treatment with IL-4 and anti-IgM. TheA1 cDNA was isolated from murine macrophages after GM-CSF induction ofdifferentiation (Lin et al., 1993). A1 is an early response gene thatdecreases its level of expression immediately following differentiationinduction (Lin et al., 1993). Yeast two-hybrid assay indicates that A1interacts strongly and selectively with Bax, with but not with any otherBcl-2 family member (Sedlak et al., 1995; Sato et al., 1994). A1 shareshomology with Bcl-2 in the BH1 and BH2 domains, but does not possess thecarboxy-terminal transmembrane domain (Lin et al., 1993).

The correlation of GM-CSF and LPS-induced differentiation with A1upregulation suggest A1 could potentially function as a cell deathsuppressor (Lin et al., 1993). Later reports has shown that A1 protectsagainst TNF induced apoptosis in the presence of actinomycin D in ahuman microvascular endothelial cells (Karsan et al., 1996). A1 couldalso inhibit ceramide induced cell death in these endothelia cells(Karsan et al., 1996). A1 expression displays a rather limited tissuedistribution and appears to be confined to hematopoietic tissues,including helper T-cells, macrophages, and neutrophils (Lin et al.,1993).

viii. Bft-1I

Bfl-1 (Bcl-2 related gene expressed in human fetal liver) was identifiedduring a random cDNA sequencing project (Choi et al., 1995). It wasfound to be homologous to Bcl-2 family members with the highest homologyto the A₁ gene. The main region of homology was in the conserved BH1,BH2, and BH3 domains. Bfl-1 is mainly expressed in bone marrow while lowlevels of expression are detected in lung, spleen, esophagus, and liver.Bfl-1 mRNA was detected at relatively high levels in six out of eightstomach cancer tumors and metastasis when compared to normal stomachtissue from the same patients (Choi et al., 1995). Bfl-1 proteinsuppresses apoptosis induced by p53 in the BRK cell line to the sameextent Bcl-2, Bcl-X_(L). Bfl-1 was also shown to cooperate with E1a inthe transformation of primary rodent epithelial cells (D'Sa-Eipper etal., 1996).

ix. GRS

GRS was incidentally cloned during the cloning of fibroblast growthfactor 4 (FGF-4) from a patient with chronic myelogenous leukemia (Lucaset al., 1994). The FGF-4 gene was truncated by a DNA rearrangement witha novel gene named GRS (Glasgow Rearranged Sequence) with a breakpoint30 nucleotides downstream from the translation termination codon ofFGF-4. The full length cDNA of GRS was then cloned from human activatedT cell cDNA library. The GRS cDNA is 824 nucleotides (Kenny et al.,1997). Sequence analysis of GRS revealed 71% identity to the murine A1protein at the amino acid level.

Northern blot analysis showed a high level of expression of GRS inhematopoietic cells and to a lesser extent in lung and kidney (Kenny etal., 1997). GRS also is expressed in cell lines of hematopoietic originincluding HL-60 (promyelocytic leukemia), Raji (Burkitt lymphoma) andK-562 (chronic myeloid leukemia). However GRS is not expressed in MOLT-4T lymphoblastic leukemia and T-cells prior to activation. The melanomacell line G-361 also expressed high levels of GRS. GRS is localized tochromosome 15q24-25. This location positions GRS adjacent to t(15;17)region translocation frequently observed in acute promyelocyticleukemia. The GRS location also places it in the breakpoint described inFanconi anemia that is associated with high incidence of acute leukemia.

x. Bid

Bid (BH3 interacting domain death agonist) was initially identified as aprotein that interacts with both Bcl-2 and Bax proteins. The labeled Baxand Bcl-2 proteins were used to screen a λEXlox expression libraryconstructed from the murine T-cell hybridoma line 2B4 (Wang et al.,1996c). Bid is a 23 kD, 195 amino acid protein. Sequence analysis of Bidrevealed that Bid shares homology only with the BH3 domain of the Bcl-2family and that it lacks the carboxy-terminus transmembrane hydrophobicdomain. A human homologue of Bid has also been identified. Human Bidshares 72.3% sequence homology to the murine Bid and has a 195 aminoacid open reading frame (Wang et al., 1996c).

In adult mouse tissue, Bid is mainly expressed in the kidneys but isalso present in brain, spleen, liver, testis and lung (Wang et al.,1996c). Low levels of expression are detected in the heart and skeletalmuscle. The mouse hematopoietic cell line, FL5.12, was also found toexpress high levels of Bid. Subcellular fractionation has revealed thatBid is predominantly localized to the cytosol (90%) with a smallfraction in the membrane fraction (Wang et al., 1996c).

Expression of Bid in the IL-3 dependent FL5.12 cell line could induce asubtle but consistent enhancement of apoptosis following IL-3 withdrawal(Wang et al., 1996c). Bid inducible expression as well as transienttransfections of Bid in Rat-1 fibroblasts and Jurkat T-cells, results inreducing cell viability to <40% at 48 h (Wang et al., 1996c). Bid couldalso restore apoptosis in FL5.12 clones overexpressing Bcl-2. The levelof apoptosis was intermediate between the parental and Bcl-2overexpressing clones. The degree of cell death in all casescorresponded to the level of Bid protein expression as detected byWestern blot analysis. Bid induced apoptosis could be inhibited byzVAD-fmk, an irreversible inhibitor particularly effective against theCPP32-like subset of proteases. This suggests that Bid induced celldeath involves activation of CPP32-like proteases (Wang et al., 1996c).

Bid interacts with both death agonists and antagonists members of theBcl-2 family. Bid can interact with. Bcl-2, Bcl-x, and Bax but does notform homodimers. Bid was unable to form trimolecular complexes withBcl-2/Bax heterodimers suggesting that Bid interacts with monomeric orhomodimeric Bcl-2 or Bax. Several mutants of Bcl-2, Bax, and Bid wereexamined to detect the regions of each molecule required for theirinteractions. The BH3 domain of Bid was essential for interaction withBax and Bcl-2. Differential specificity of these mutants was alsodetected as mutant (M97A, D98A) could bind Bax but not Bcl-2, mutant(G97A) could bind Bcl-2 but not Bax while other mutants did not bindeither protein. Noteworthy is that all BH3 mutants of Bid were impairedin their ability to counter Bcl-2 protection including mutants thatcould still bind Bcl-2. However, Bid mutant (M97A, D98A) that can stillbind Bax but not Bcl-2, retained its activity. Conversely, the BH1domain of Bcl-2 and Bax were shown to be required for their interactionwith Bid. It is suggested that the a helix BH3 domain of Bid interactswith the hydrophobic cleft contributed by the BH1 domain of Bcl-x. Thisinteraction might result in a conformational change in Bid, Bcl-2, orBax that signals cell death.

xi. Bik

Bik (Bcl-2 interacting killer) is a novel Bcl-2 family member that wasdetected when a human B-cell line cDNA library was used in a yeast twohybrid screen for proteins that interact with Bcl-2 (Boyd et al., 1995).Bik is a 160 amino acid protein and has a predicted molecular weight of18 kD encoded by 928 bp cDNA and 1 Kb mRNA. Bik shares homology onlywithin the BH3 domain of the Bcl-2 family and has a carboxy-terminaltransmembrane hydrophobic domain. Bik was found to localize to thenuclear envelope and cytoplasmic membrane structures.

Transient co-transfection of Bik and β-galactosidase expression plasmidsin Rat-1 fibroblasts resulted in a dramatic reduction in the number ofblue cells, consistent with reduced viability of Bik transfected cells(Boyd et al., 1995). Co-transfection of Bik and Bcl-2, Bcl-x, adenovirusE1B-19 kDa, or EBV-BHRF1 resulted in an increase in blue cell numberindicating the ability of these proteins to reverse cell death by Bik.Deletion of the BH3 domain of Bik resulted in loss of its pro-apoptoticactivity. Bik induced apoptosis was shown to be inhibited by zVAD-fmk.However, CrmA could not inhibit Bik induced cell death. This suggeststhat Bik induced cell death involves selective activation of CPP32-likeproteases (Orth and Dixit, 1997).

Interactions between Bik and other Bcl-2 family members was examinedusing the yeast two hybrid system, GST-fusion protein capture onglutathione agarose beads, and transient co-transfection of tagged Bikwith other anti-apoptotic Bcl-2 family members (Boyd et al., 1995).These in vitro and in vivo studies revealed interactions between Bik andBcl-2, Bcl-x, adenovirus E1B-19 kDa, and EBV-BHRF1. Bik also interactswith Bcl-x, a death promoting protein that lacks BH1 and BH2 domains.This suggests that Bik does not require BH1 and BH2 domain for itsinteraction with Bcl-2 family members. Bcl-2 residues 43-48 and E1B-19kDa residues 90-96 were shown to be essential for interaction with Bik.Noteworthy is that these residues are not within the conserved regionsof Bcl-2 family members.

xii. Bcl-w

Bcl-w was cloned using degenerate primers to the conserved BH1 and BH2domains in a low stringency PCR™ reaction (Gibson et al., 1996). Theseprimers were used to amplify cDNA from mouse macrophage and mouse braincell lines. The PCR™ product was then used to screen cDNA libraries frommouse brain, spleen, and myeloid cell lines. Bcl-w is a 22 Kb gene witha 3.7 Kb mRNA which encodes a 22 kD protein. Human Bcl-w was thenisolated from an adult human brain cDNA library. Bcl-w possesses theBH1, BH2, and BH3 domains. The human and mouse genes are 99% identicalat the amino acid level and 94% at the nucleotide level.

Bcl-w mRNA is expressed at high levels in brain, colon, and salivarygland. Surprisingly, Bcl-w expression is not detected in T- andB-lymphoid cell lines. However, mRNA was detected in myeloid cell linesof macrophage, megakaryocyte, erythroid, and mast cell origin. Bcl-walso has a hydrophobic C-terminal transmembrane domain. The cytoplasmiclocalization of Bcl-w is similar to that of Bcl-2. Bcl-w resides in thecentral region of mouse chromosome 14 and human chromosome 14 at q11.2.Hematopoetic cell lines expressing Bcl-w were resistant to apoptosisinduction to the same extent as Bcl-2 and Bcl-x stable transfectants.However, Bcl-w did not protect CHI B-lymphoma cells from CD95-inducedapoptosis while Bcl-2 and Bcl-x_(L) were able to do so (Gibson et al.,1996).

xiii. Harakiri

The Harakiri gene and its protein product Hrk was identified by a yeasttwo hybrid screen of a HeLA cDNA library to detect proteins that bind toBcl-2 (Inohara et al., 1997). A 9-wk human embryo cDNA library was usedto obtain the full length Hrk cDNA. Hrk was detected as a 716 bp cDNAthat was confirmed by the northern blot analysis using both human andmouse tissue as 0.7 Kb mRNA. The cDNA encodes an open reading flame of91 amino acids. Hrk shares homology with Bcl-2 family member BH3 domain,however, the rest of the protein has no significant homology to anyother protein or Bcl-2 family. A region of 28 hydrophobic amino acidsthat may serve as a membrane-spanning domain was also identified at theCOOH-terminus of Hrk.

Northern blot analysis demonstrates high levels of Hrk expression in alllymphoid tissues examined including the bone marrow and spleen. Hrk isalso expressed in the pancreas and at low levels in the kidney, liver,lung, and brain (Inohara et al., 1997). Hrk was seen as a cytosolicgranular staining by confocal microscopy of transiently transfectedcells with flagged Hrk. This staining is similar to the previouslyreported localization of Bcl-2 and Bcl-x.

Transient transfections of Harakiri in 293T cells, HeLa, and FL5.12progenitor B-cells resulted in a dramatic decrease in cell viability by36 h post-transfection. However co-expression of Bcl-2 and Bcl-x couldinhibit the death promoting activity of Hrk. Interestingly, Hrk appearsto interact only with Bcl-2 and Bcl-x_(L) but not with the otherpro-apoptotic family members Bax, Bak, and Bcl-x_(s). Deletion mutantsof Hrk lacking 16 amino acids including the BH3 domain were unable tointeract with Bcl-2 and Bcl-x. This mutant also failed to induce celldeath in 293T cells. Deletion analysis has also revealed the requirementof BH1 and BH2 domains of Bcl-2 and Bcl-x to interact with Hrk.

C. Interactions of Bcl-2 Family Members and Mechanisms of Function

One of the reasons for the modest understanding of the mechanisms bywhich Bcl-2 homologues execute their cellular roles stems from a lack ofidentifiable sequence motifs in the Bcl-2 family which would implicate amechanism of action. What have been defined, however, are shared domainsdesignated as Bcl-2 homology domain 1, 2, 3 and 4. The BH1 domain spansamino acid residues 136-155 of the Bcl-2 protein, BH2 spans resides187-202, BH3 spans resides 93-107 and BH4 spans residues 10-30. The BH3domain, for its pan appears to be involved in selective interactionsbetween Bcl-2 family members.

The BH3 domain appears to be required for the death promoting activityof Bax and bak are required for their interaction with twodeath-repressing members, Bcl-2 and Bcl-x_(L) (Zha et al., 1996a;Chittenden et al., 1995).

The BH1 and BH2 domains serve equally important functions. The creationof point mutations in either domain, can effectively abolish the deathrepression function of Bcl-2 (Yin et al., 1994). Recent evidencesuggests, however, that the formation of heterodimers is not requiredfor function of family members (Cheng et al., 1996). These same BH1 andBH2 domain mutants of Bcl-2 fail to heterodimerize with Bax, althoughthey do homodimerize well (Yin et al., 1994). Some of the mostcompelling evidence that the BH3 motif represents a “death domain” comesfrom studies of Bid (Wang et al., 1996c). Bid possesses only the BH3domain, lacks the carboxy-terminal signal-anchor segment, and localizesto both cytosolic and membrane compartments. Importantly, ectopicexpression of Bid abrogates the pro-survival effect of Bcl-2.Additionally, expression of Bid, without another death stimulus, inducesICE-like proteases and apoptosis. An intact BH3 domain of Bid wasrequired to bind the BH1 domain of either Bcl-2 or Bax.

The BH4 domain, which is located at the amino-terminus has been far lesscharacterized. To date, it has been reported that deletion of the BH4domain of Bcl-2 nullifies anti-apoptotic function and homodimerization,but does not impair Bcl-2/Bax heterodimerization (Reed et al., 1996).There is some evidence which indicates that the BH4 domain may mediateinteractions of Bcl-2 family member protein with non-Bcl-2-relatedproteins such as calcineurin (Shibasaki et al., 1997). Thus the BH4domain may serve as an tethering domain that bridges Bcl-2 andBcl-2-related proteins to important signal transduction proteins.

Perhaps, at its simplest level, the expression of various Bcl-2 relatedproteins may determine whether a cell responds to an applied stress byinitiating a cell death program or surviving. However, anotherhypothesis, that has substantial experimental evidence based on amutational analysis of the BH domains, suggests that the cellularresponse to injury may be a function of the multiple heterodimerizationand homodimerization states between members of this protein family. Thismodel, commonly known as the “rheostat model” has been advocated by Dr.Stanley Korsmeyer's group (Oltvai et al., 1993; Korsmeyer et al., 1993).In this scenario, the relative levels of dimerization partners availableshifts the balance of cell fate in favor of viability (e.g., Bcl-2/Bcl-2homodimers favoring cell survival) or cell death (e.g., Bax/Baxhomodimers favoring cell death) following exposure to an appropriatestress. This ability of Bcl-2-related proteins to hereto- andhomodimerize in vivo, is perhaps one of the most important features ofthe family.

Complicating the picture further are reports of the ability of severalBcl-2 family members to physically interact with several signalingprotein complexes containing p21 ras (Chen and Faller, 1996), Raf-1kinase (Wang et al., 1996b) and p23 R-ras proteins (Wang et al., 1995).Another feature, is the conservation of a hydrophobic membrane targetingsequence in the carboxy-terminal tail of most members of the Bcl-2family. The targeting domain most likely ensures that the variousmembers are correctly routed to the appropriate intracellular organelle.Perhaps, this routing domain ensures that the various Bcl-2-relatedproteins are localized in close proximity to secure proper physicalinteractions should the appropriate stress be detected.

The mechanisms of programmed cell death are far from being completelyelucidated. At present, many different factors such as proteaseactivation (Yuan, et al., 1993; Fraser and Evan, et al., 1996;Chinnaiyan et al., 1997), DNA cleavage, and calcium signaling (Lam etal., 1994; Marin et al., 1996; Minn et al., 1997) are known toparticipate in apoptosis. The placement of Bcl-2 and Bcl-2 familymembers in cell death regulatory pathways is now being elucidated. It isnow known that Bcl-x_(L) can form ion channels and it may be that otherBcl-2 family members function in a similar manner. The specificinteractions that Bcl-2 family proteins have with various signalingmolecules and within the Bcl-2 family itself are active areas ofinvestigation.

D. Engineering Expression Constructs

In certain embodiments, the present invention involves the manipulationof genetic material to produce expression constructs that encodetherapeutic genes. Such methods involve the generation of expressionconstructs containing, for example, a heterologous DNA encoding a geneof interest and a means for its expression, replicating the vector in anappropriate helper cell, obtaining viral particles produced therefrom,and infecting cells with the recombinant virus particles.

The gene will be a therapeutic gene such as one or more of theproapoptotic genes discussed herein above, or the gene may be a secondtherapeutic gene or nucleic acid useful in the treatment of, for examplecancer cells. In the context of gene therapy, the gene will be aheterologous DNA, meant to include DNA derived from a source other thanthe viral genome which provides the backbone of the vector. Finally, thevirus may act as a live viral vaccine and express an antigen of interestfor the production of antibodies thereagainst. The gene may be derivedfrom a prokaryotic or eukaryotic source such as a bacterium, a virus, ayeast, a parasite, a plant, or even an animal. The heterologous DNA alsomay be derived from more than one source, i.e., a multigene construct ora fusion protein. The heterologous DNA also may include a regulatorysequence which may be derived from one source and the gene from adifferent source.

i. Additional Therapeutic Genes

The present invention contemplates the use of a variety of differentgenes in combination with adenoviral Bax and the proapoptotic Bcl-2 geneconstructs. For example, genes encoding enzymes, hormones, cytokines,oncogenes, receptors, tumor suppressors, transcription factors, drugselectable markers, toxins and various antigens are contemplated assuitable genes for use according to the present invention. In addition,antisense constructs derived from oncogenes are other “genes” ofinterest according to the present invention.

a. p53

As stated earlier, p53 currently is recognized as a tumor suppressorgene. High levels of mutant p53 have been found in many cellstransformed by chemical carcinogenesis, ultraviolet radiation, andseveral viruses. The p53 gene is a frequent target of mutationalinactivation in a wide variety of human tumors and is already documentedto be the most frequently-mutated gene in common human cancers. It ismutated in over 50% of human NSCLC (Hollstein et al., 1991) and in awide spectrum of other tumors.

The p53 gene encodes a 393-amino acid phosphoprotein that can formcomplexes with host proteins such as large-T antigen and E1B. Theprotein is found in normal tissues and cells, but at concentrationswhich are minute by comparison with transformed cells or tumor tissue.Interestingly, wild-type p53 appears to be important in regulating cellgrowth and division. Overexpression of wild-type p53 has been shown insome cases to be anti-proliferative in human tumor cell lines. Thus, p53can act as a negative regulator of cell growth (Weinberg, 1991) and maydirectly suppress uncontrolled cell growth or indirectly activate genesthat suppress this growth. Thus, absence or inactivation of wild-typep53 may contribute to transformation. However, some studies indicatethat the presence of mutant p53 may be necessary for full expression ofthe transforming potential.

Wild-type p53 is recognized as an important growth regulator in manycell types. Missense mutations are common for the p53 gene and areessential for the transforming ability of the oncogene. A single geneticchange prompted by point mutations can create carcinogenic p53. Unlikeother oncogenes, however, p53 point mutations are known to occur in atleast 30 distinct codons, often creating dominant alleles that produceshifts in cell phenotype without a reduction to homozygosity.Additionally, many of these dominant negative alleles appear to betolerated in the organism and passed on in the germ line. Various mutantalleles appear to range from minimally dysfunctional to stronglypenetrant, dominant negative alleles (Weinberg, 1991).

Casey and colleagues have reported that transfection of DNA encodingwild-type p53 into two human breast cancer cell lines restores growthsuppression control in such cells (Casey et al., 1991). A similar effecthas also been demonstrated on transfection of wild-type, but not mutant,p53 into human lung cancer cell lines (Takahasi et al., 1992). p53appears dominant over the mutant gene and will select againstproliferation when transfected into cells with the mutant gene. Normalexpression of the transfected p53 does not affect the growth of cellswith endogenous p53. Thus, such constructs might be taken up by normalcells without adverse effects. It is thus proposed that the treatment ofp53-associated cancers with wild-type p53 or other therapies describedherein will reduce the number of malignant cells or their growth rate,alternatively the treatment will result in the decrease of themetastatic potential of the cancer cell, a decrease in tumor size or ahalt in the growth the tumor.

b. p16

The major transitions of the eukaryotic cell cycle are triggered bycyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent kinase 4(CDK4), regulates progression through the G₁. The activity of thisenzyme may be to phosphorylate Rb at late G₁. The activity of CDK4 iscontrolled by an activating subunit, D-type cyclin, and by an inhibitorysubunit, the p16^(INK4) has been biochemically characterized as aprotein that specifically binds to and inhibits CDK4, and thus mayregulate Rb phosphorylation (Serrano et al., 1993; Serrano et al.,1995). Since the p16^(INK4) protein is a CDK4 inhibitor (Serrano, 1993),deletion of this gene may increase the activity of CDK4, resulting inhyperphosphorylation of the Rb protein. p16 also is known to regulatethe function of CDK6.

p16^(INK4) belongs to a newly described class of CDK-inhibitory proteinsthat also includes p16^(B), p21^(WAF1), and p27^(KIP1). The p16^(INK4)gene maps to 9p21, a chromosome region frequently deleted in many tumortypes. Homozygous deletions and mutations of the p16^(INK4) gene arefrequent in human tumor cell lines. This evidence suggests that thep16^(INK4) gene is a tumor suppressor gene. This interpretation has beenchallenged, however, by the observation that the frequency of thep16^(INK4) gene alterations is much lower in primary uncultured tumorsthan in cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori etal., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et al.,1994; Arap et al., 1995). Restoration of wild-type p16^(INK4) functionby transfection with a plasmid expression vector reduced colonyformation by some human cancer cell lines (Okamoto, 1994; Arap, 1995).

c. C-CAM

C-CAM is expressed in virtually all epithelial cells (Odin and Obrink,1987). C-CAM, with an apparent molecular weight of 105 kD, wasoriginally isolated from the plasma membrane of the rat hepatocyte byits reaction with specific antibodies that neutralize cell aggregation(Obrink, 1991). Recent studies indicate that, structurally, C-CAMbelongs to the immunoglobulin (Ig) superfamily and its sequence ishighly homologous to carcinoembryonic antigen (CEA) (Lin and Guidotti,1989). Using a baculovirus expression system, Cheung et al. (1993)demonstrated that the first Ig domain of C-CAM is critical for celladhesive activity.

Cell adhesion molecules, or CAM's are known to be involved in a complexnetwork of molecular interactions that regulate organ development andcell differentiation (Edelman, 1985). Recent data indicate that aberrantexpression of CAM's maybe involved in the tumorigenesis of severalneoplasms; for example, decreased expression of E-cadherin, which ispredominantly expressed in epithelial cells, is associated with theprogression of several kinds of neoplasms (Edelman and Crossin, 1991;Frixen et al., 1991; Bussemakers et al., 1992; Matsura et al., 1992;Umbas et al., 1992). Also, Giancotti and Ruoslahti (1990) demonstratedthat increasing expression of α₅β₁ integrin by gene transfer can reducetumorigenicity of Chinese hamster ovary cells in vivo. C-CAM now hasbeen shown to suppress tumors growth in vitro and in vivo.

d. Other Tumor Suppressors

Other tumor suppressors that may be employed according to the presentinvention include RB, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II, zac1,p73, VHL, MMAC1, FCC and MCC. Additional inducers of apoptosis inaddition to those of the Bcl-2 family, such as, Ad E1B and ICE-CED3proteases, similarly could find use according to the present invention.

e. Enzymes

Various enzyme genes are of interest according to the present invention.Such enzymes include cytosine deaminase, hypoxanthine-guaninephosphoribosyltransferase, galactose-1-phosphate uridyltransferase,phenylalanine hydroxylase, glucocerbrosidase, sphingomyelinase,α-L-iduronidase, glucose-6-phosphate dehydrogenase, HSV thymidine kinaseand human thymidine kinase.

f. Cytokines

Other classes of genes that are contemplated to be inserted into thetherapeutic expression constructs of the present invention includeinterleukins and cytokines. Interleukin 1 (IL-1), IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.

g. Antibodies

In yet another embodiment, the heterologous gene may include asingle-chain antibody. Methods for the production of single-chainantibodies are well known to those of skill in the art. The skilledartisan is referred to U.S. Pat. No. 5,359,046, (incorporated herein byreference) for such methods. A single chain antibody is created byfusing together the variable domains of the heavy and light chains usinga short peptide linker, thereby reconstituting an antigen binding siteon a single molecule.

Single-chain antibody variable fragments (Fvs) in which the C-terminusof one variable domain is tethered to the N-terminus of the other via a15 to 25 amino acid peptide or linker, have been developed withoutsignificantly disrupting antigen binding or specificity of the binding(Bedzyk et al., 1990; Chaudhary et al., 1990). These Fvs lack theconstant regions (Fc) present in the heavy and light chains of thenative antibody.

Antibodies to a wide variety of molecules can be used in combinationwith the present invention, including antibodies against oncogenes,toxins, hormones, enzymes, viral or bacterial antigens, transcriptionfactors, receptors and the like.

ii. Antisense Constructs

Oncogenes such as ras, myc, neu, raf erb, src, fms, jun, trk, ret, gsp,hst, and abl as well as the antiapoptotic member of the Bcl-2 familyalso are suitable targets. However, for therapeutic benefit, theseoncogenes would be expressed as an antisense nucleic acid, so as toinhibit the expression of the oncogene. The term “antisense nucleicacid” is intended to refer to the oligonucleotides complementary to thebase sequences of oncogene-encoding DNA and RNA. Antisenseoligonucleotides, when introduced into a target cell, specifically bindto their target nucleic acid and interfere with transcription, RNAprocessing, transport and/or translation. Targeting double-stranded (ds)DNA with oligonucleotide leads to triple-helix formation; targeting RNAwill lead to double-helix formation.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. Antisense RNA constructs, or DNA encoding such antisense RNAs, maybe employed to inhibit gene transcription or translation or both withina host cell, either in vitro or in vivo, such as within a host animal,including a human subject. Nucleic acid sequences comprising“complementary nucleotides” are those which are capable of base-pairingaccording to the standard Watson-Crick complementary rules. That is,that the larger purines will base pair with the smaller pyrimidines toform only combinations of guanine paired with cytosine (G:C) and adeninepaired with either thymine (A:T), in the case of DNA, or adenine pairedwith uracil (A:U) in the case of RNA.

As used herein, the terms “complementary” or “antisense sequences” meannucleic acid sequences that are substantially complementary over theirentire length and have very few base mismatches. For example, nucleicacid sequences of fifteen bases in length may be termed complementarywhen they have a complementary nucleotide at thirteen or fourteenpositions with only single or double mismatches. Naturally, nucleic acidsequences which are “completely complementary” will be nucleic acidsequences which are entirely complementary throughout their entirelength and have no base mismatches.

While all or part of the gene sequence may be employed in the context ofantisense construction, statistically, any sequence 17 bases long shouldoccur only once in the human genome and, therefore, suffice to specify aunique target sequence. Although shorter oligomers are easier to makeand increase in vivo accessibility, numerous other factors are involvedin determining the specificity of hybridization. Both binding affinityand sequence specificity of an oligonucleotide to its complementarytarget increases with increasing length. It is contemplated thatoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore base pairs will be used. One can readily determine whether a givenantisense nucleic acid is effective at targeting of the correspondinghost cell gene simply by testing the constructs in vitro to determinewhether the endogenous gene's function is affected or whether theexpression of related genes having complementary sequences is affected.

In certain embodiments, one may wish to employ antisense constructswhich include other elements, for example, those which include C-5propyne pyrimidines. Oligonucleotides which contain C-5 propyneanalogues of uridine and cytidine have been shown to bind RNA with highaffinity and to be potent antisense inhibitors of gene expression(Wagner et al., 1993).

iii. Ribozyme Constructs

As an alternative to targeted antisense delivery, targeted ribozymes maybe used. The term “ribozyme” refers to an RNA-based enzyme capable oftargeting and cleaving particular base sequences in oncogene DNA andRNA. Ribozymes either can be targeted directly to cells, in the form ofRNA oligo-nucleotides incorporating ribozyme sequences, or introducedinto the cell as an expression construct encoding the desired ribozymalRNA. Ribozymes may be used and applied in much the same way as describedfor antisense nucleic acids.

iv. Selectable Markers

In certain embodiments of the invention, the therapeutic expressionconstructs of the present invention contain nucleic acid constructswhose expression may be identified in vitro or in vivo by including amarker in the expression construct. Such markers would confer anidentifiable change to the cell permitting easy identification of cellscontaining the expression construct. Usually the inclusion of a drugselection marker aids in cloning and in the selection of transformants.For example, genes that confer resistance to neomycin, puromycin,hygromycin, DHFR, GPT, zeocin and histidinol are useful selectablemarkers. Alternatively, enzymes such as herpes simplex virus thymidinekinase (tk) may be employed. Immunologic markers also can be employed.The selectable marker employed is not believed to be important, so longas it is capable of being expressed simultaneously with the nucleic acidencoding a gene product. Further examples of selectable markers are wellknown to one of skill in the art and include reporters such as EGFP,β-gal or chloramphenicol acetyltransferase (CAT).

v. Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosomebinding sites (IRES) elements are used to create multigene polycistronicmessages. IRES elements are able to bypass the ribosome scanning modelof 5′-methylated, Cap-dependent translation and begin translation atinternal sites (Pelletier and Sonenberg, 1988). IRES elements from twomembers of the picanovirus family (polio and encephalomyocarditis) havebeen described (Pelletier and Sonenberg, 1988), as well an IRES from amammalian message (Macejak and Sarnow, 1991). IRES elements can belinked to heterologous open reading frames. Multiple open reading framescan be transcribed together, each separated by an IRES, creatingpolycistronic messages. By virtue of the IRES element, each open readingframe is accessible to ribosomes for efficient translation. Multiplegenes can be efficiently expressed using a single promoter/enhancer totranscribe a single message.

Any heterologous open reading frame can be linked to IRES elements. Thisincludes genes for secreted proteins, multi-subunit proteins, encoded byindependent genes, intracellular or membrane-bound proteins andselectable markers. In this way, expression of several proteins can besimultaneously engineered into a cell with a single construct and asingle selectable marker.

vi. Control Regions

A. Promoters

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor gene products in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding genes of interest.

The nucleic acid encoding a gene product is under transcriptionalcontrol of a promoter. A “promoter” refers to a DNA sequence recognizedby the machinery of the cell, or introduced machinery, required toinitiate the specific transcription of a gene. The phrase “undertranscriptional control” means that the promoter is in the correctlocation and orientation in relation to the nucleic acid to control RNApolymerase initiation and expression of the gene.

The term promoter will be used here to refer to a group oftranscriptional control modules that are clustered around the initiationsite for RNA polymerase II. Much of the thinking about how promoters areorganized derives from analyses of several viral promoters, includingthose for the HSV thymidine kinase (tk) and SV40 early transcriptionunits. These studies, augmented by more recent work, have shown thatpromoters are composed of discrete functional modules, each consistingof approximately 7-20 bp of DNA, and containing one or more recognitionsites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either co-operatively or independently to activatetranscription.

The particular promoter employed to control the expression of a nucleicacid sequence of interest is not believed to be important, so long as itis capable of directing the expression of the nucleic acid in thetargeted cell. Thus, where a human cell is targeted, it is preferable toposition the nucleic acid coding region adjacent to and under thecontrol of a promoter that is capable of being expressed in a humancell. Generally speaking, such a promoter might include either a humanor viral promoter.

In various embodiments, the human cytomegalovirus (CMV) immediate earlygene promoter, the SV40 early promoter, the Rous sarcoma virus longterminal repeat, β-actin, rat insulin promoter andglyceraldehyde-3-phosphate dehydrogenase can be used to obtainhigh-level expression of the coding sequence of interest. The use ofother viral or mammalian cellular or bacterial phage promoters which arewell-known in the art to achieve expression of a coding sequence ofinterest is contemplated as well, provided that the levels of expressionare sufficient for a given purpose. By employing a promoter withwell-known properties, the level and pattern of expression of theprotein of interest following transfection or transformation can beoptimized.

Selection of a promoter that is regulated in response to specificphysiologic or synthetic signals can permit inducible expression of thegene product. For example in the case where expression of a transgene,or transgenes when a multicistronic vector is utilized, is toxic to thecells in which the vector is produced in, it may be desirable toprohibit or reduce expression of one or more of the transgenes. Examplesof transgenes that may be toxic to the producer cell line arepro-apoptotic and cytokine genes. Several inducible promoter systems areavailable for production of viral vectors where the transgene productmay be toxic.

The ecdysone system (Invitrogen, Carlsbad, Calif.) is one such system.This system is designed to allow regulated expression of a gene ofinterest in mammalian cells. It consists of a tightly regulatedexpression mechanism that allows virtually no basal level expression ofthe transgene, but over 200-fold inducibility. The system is based onthe heterodimeric ecdysone receptor of Drosophila, and when ecdysone oran analog such as muristerone A binds to the receptor, the receptoractivates a promoter to turn on expression of the downstream transgenehigh levels of mRNA transcripts are attained. In this system, bothmonomers of the heterodimeric receptor are constituitively expressedfrom one vector, whereas the ecdysone-responsive promoter which drivesexpression of the gene of interest is on another plasmid. Engineering ofthis type of system into the gene transfer vector of interest wouldtherefore be useful. Cotransfection of plasmids containing the gene ofinterest and the receptor monomers in the producer cell line would thenallow for the production of the gene transfer vector without expressionof a potentially toxic transgene. At the appropriate time, expression ofthe transgene could be activated with ecdysone or muristeron A.

Another inducible system that would be useful is the Tet-Off™ or Tet-On™system (Clontech, Palo Alto, Calif.) originally developed by Gossen andBujard (Gossen and Bujard, 1992; Gossen et al., 1995). This system alsoallows high levels of gene expression to be regulated in response totetracycline or tetracycline derivatives such as doxycycline. In theTet-On™ system, gene expression is turned on in the presence ofdoxycycline, whereas in the Tet-Off™ system, gene expression is turnedon in the absence of doxycycline. These systems are based on tworegulatory elements derived from the tetracycline resistance operon ofE. coli. The tetracycline operator sequence to which the tetracyclinerepressor binds, and the tetracycline repressor protein. The gene ofinterest is cloned into a plasmid behind a promoter that hastetracycline-responsive elements present in it. A second plasmidcontains a regulatory element called the tetracycline-controlledtransactivator, which is composed, in the Tet-Off™ system, of the VP16domain from the herpes simplex virus and the wild-type tertracyclinerepressor. Thus in the absence of doxycycline, transcription isconstituitively on. In the Tet-On™ system, the tetracycline repressor isnot wild type and in the presence of doxycycline activatestranscription. For gene therapy vector production, the Tet-Off™ systemwould be preferable so that the producer cells could be grown in thepresence of tetracycline or doxycycline and prevent expression of apotentially toxic transgene, but when the vector is introduced to thepatient, the gene expression would be constituitively on.

In some circumstances, it may be desirable to regulate expression of atransgene in a gene therapy vector. For example, different viralpromoters with varying strengths of activity may be utilized dependingon the level of expression desired. In mammalian cells, the CMVimmediate early promoter if often used to provide strong transcriptionalactivation. Modified versions of the CMV promoter that are less potenthave also been used when reduced levels of expression of the transgeneare desired. When expression of a transgene in hematopoetic cells isdesired, retroviral promoters such as the LTRs from MLV or MMTV areoften used. Other viral promoters that may be used depending on thedesired effect include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenoviruspromoters such as from the E1A, E2A, or MLP region, AAV LTR, cauliflowermosaic virus, HSV-TK, and avian sarcoma virus.

Similarly tissue specific promoters may be used to effect transcriptionin specific tissues or cells so as to reduce potential toxicity orundesirable effects to non-targeted tissues. For example, promoters suchas the PSA, probasin, prostatic acid phosphatase or prostate-specificglandular kallikrein (hK2) may be used to target gene expression in theprostate. Similarly, the following promoters may be used to target geneexpression in other tissues (Table 2). TABLE 2 Tissue specific promotersTissue Promoter Pancreas insulin elastin amylase pdr-1 pdx-1 glucokinaseLiver albumin PEPCK HBV enhancer alpha fetoprotein apolipoprotein Calpha-1 antitrypsin vitellogenin, NF-AB Transthyretin Skeletal musclemyosin H chain muscle creatine kinase dystrophin calpain p94 skeletalalpha-actin fast troponin 1 Skin keratin K6 keratin K1 Lung CFTR humancytokeratin 18 (K18) pulmonary surfactant proteins A, B and C CC-10 P1Smooth muscle sm22 alpha SM-alpha-actin Endothelium endothelin-1E-selectin von Willebrand factor TIE (Korhonen et al., 1995) KDR/flk-1Melanocytes tyrosinase Adipose tissue lipoprotein lipase (Zechner etal., 1988) adipsin (Spiegelman et al., 1989) acetyl-CoA carboxylase(Pape and Kim, 1989) glycerophosphate dehydrogenase (Dani et al., 1989)adipocyte P2 (Hunt et al., 1986) Blood β-globin

In certain indications, it may be desirable to activate transcription atspecific times after administration of the gene therapy vector. This maybe done with such promoters as those that are hormone or cytokineregulatable. For example in gene therapy applications where theindication is a gonadal tissue where specific steroids are produced orrouted to, use of androgen or estrogen regulated promoters may beadvantageous. Such promoters that are hormone regulatable include MMTV,MT-1, ecdysone and RuBisco. Other hormone regulated promoters such asthose responsive to thyroid, pituitary and adrenal hormones are expectedto be useful in the present invention. Cytokine and inflammatory proteinresponsive promoters that could be used include K and T Kininogen(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein (Arcone etal., 1988), haptoglobin (Oliviero et al., 1987), serum amyloid A2, C/EBPalpha, IL-1, IL-6 (Poli and Cortese, 1989), Complement C3 (Wilson etal., 1990), IL-8, alpha-1 acid glycoprotein (Prowse and Baumann, 1988),alpha-1 antitypsin, lipoprotein lipase (Zechner et al., 1988),angiotensinogen (Ron et al., 1991), fibrinogen, c-jun (inducible byphorbol esters, TNF-alpha, UV radiation, retinoic acid, and hydrogenperoxide), collagenase (induced by phorbol esters and retinoic acid),metallothionein (heavy metal and glucocorticoid inducible), Stromelysin(inducible by phorbol ester, interleukin-1 and EGF), alpha-2macroglobulin and alpha-1 antichymotrypsin.

It is envisioned that cell cycle regulatable promoters may be useful inthe present invention. For example, in a bi-cistronic gene therapyvector, use of a strong CMV promoter to drive expression of a first genesuch as p16 that arrests cells in the G1 phase could be followed byexpression of a second gene such as p53 under the control of a promoterthat is active in the G1 phase of the cell cycle, thus providing a“second hit” that would push the cell into apoptosis. Other promoterssuch as those of various cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1could be used.

Tumor specific promoters such as osteocalcin, hypoxia-responsive element(HRE), MAGE-4, CEA, alpha-fetoprotein, GRP78/BiP and tyrosinase may alsobe used to regulate gene expression in tumor cells. Other promoters thatcould be used according to the present invention includeLac-regulatable, chemotherapy inducible (e.g. MDR), and heat(hyperthermia) inducible promoters, Radiation-inducible (e.g., EGR (Jokiet al., 1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acidpromoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, -actin andalpha-globin. Many other promoters that may be useful are listed inWalther and Stein (1996).

It is envisioned that any of the above promoters alone or in combinationwith another may be useful according to the present invention dependingon the action desired. In addition, this list of promoters is should notbe construed to be exhaustive or limiting, those of skill in the artwill know of other promoters that may be used in conjunction with thepromoters and methods disclosed herein.

B. Enhancers

Enhancers are genetic elements that increase transcription from apromoter located at a distant position on the same molecule of DNA.Enhancers are organized much like promoters. That is, they are composedof many individual elements, each of which binds to one or moretranscriptional proteins. The basic distinction between enhancers andpromoters is operational. An enhancer region as a whole must be able tostimulate transcription at a distance; this need not be true of apromoter region or its component elements. On the other hand, a promotermust have one or more elements that direct initiation of RNA synthesisat a particular site and in a particular orientation, whereas enhancerslack these specificities. Promoters and enhancers are often overlappingand contiguous, often seeming to have a very similar modularorganization.

Below is a list of promoters additional to the tissue specific promoterslisted above, cellular promoters/enhancers and induciblepromoters/enhancers that could be used in combination with the nucleicacid encoding a gene of interest in an expression construct (Table 3 andTable 4). Additionally, any promoter/enhancer combination (as per theEukaryotic Promoter Data Base EPDB) could also be used to driveexpression of the gene. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct. TABLE 3 ENHANCERImmunoglobulin Heavy Chain Immunoglobulin Light Chain T-Cell ReceptorHLA DQ α and DQ β β-Interferon Interleukin-2 Interleukin-2 Receptor MHCClass II 5 MHC Class II HLA-DRα β-Actin Muscle Creatine KinasePrealbumin (Transthyretin) Elastase I Metallothionein CollagenaseAlbumin Gene α-Fetoprotein τ-Globin β-Globin e-fos c-HA-ras InsulinNeural Cell Adhesion Molecule (NCAM) α1-Antitrypsin H2B (TH2B) HistoneMouse or Type I Collagen Glucose-RegulatedProteins (GRP94 and GRP78) RatGrowth Hormone Human Serum Amyloid A (SAA) Troponin I (TN I)Platelet-Derived Growth Factor Duchenne Muscular Dystrophy SV40 PolyomaRetroviruses Papilloma Virus Hepatitis B Virus Human ImmunodeficiencyVirus Cytomegalovirus Gibbon Ape Leukemia Virus

TABLE 4 Element Inducer MT II Phorbol Ester (TPA) Heavy metals MMTV(mouse mammary tumor Glucocorticoids virus) β-Interferon poly(rI)Xpoly(rc) Adenovirus 5 E2 Ela c-jun Phorbol Ester (TPA), H₂O₂ CollagenasePhorbol Ester (TPA) Stromelysin Phorbol Ester (TPA), IL-1 SV40 PhorbolEster (TPA) Murine MX Gene Interferon, Newcastle Disease Virus GRP78Gene A23187 α-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene H-2kBInterferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol Ester-TPATumor Necrosis Factor FMA Thyroid Stimulating Hormone α Thyroid HormoneGene Insulin E Box Glucose

In preferred embodiments of the invention, the expression constructcomprises a virus or engineered construct derived from a viral genome.The ability of certain viruses to enter cells via receptor-mediatedendocytosis and to integrate into host cell genome and express viralgenes stably and efficiently have made them attractive candidates forthe transfer of foreign genes into mammalian cells (Ridgeway, 1988;Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986).The first viruses used as gene vectors were DNA viruses including thepapovaviruses (simian virus 40, bovine papilloma virus, and polyoma)(Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway,1988; Baichwal and Sugden, 1986). These have a relatively low capacityfor foreign DNA sequences and have a restricted host spectrum.Furthermore, their oncogenic potential and cytopathic effects inpermissive cells raise safety concerns. They can accommodate only up to8 kB of foreign genetic material but can be readily introduced in avariety of cell lines and laboratory animals (Nicolas and Rubenstein,1988; Temin, 1986).

C. Polyadenylation Signals

Where a cDNA insert is employed, one will typically desire to include apolyadenylation signal to effect proper polyadenylation of the genetranscript. The nature of the polyadenylation signal is not believed tobe crucial to the successful practice of the invention, and any suchsequence may be employed such as human or bovine growth hormone and SV40polyadenylation signals. Also contemplated as an element of theexpression cassette is a terminator. These elements can serve to enhancemessage levels and to minimize read through from the cassette into othersequences.

E. Methods of Gene Transfer

In order to mediate the effect transgene expression in a cell, it willbe necessary to transfer the therapeutic expression constructs of thepresent invention into a cell. Such transfer may employ viral ornon-viral methods of gene transfer. This section provides a discussionof methods and compositions of gene transfer.

i. Viral Vector-Mediated Transfer

The proapoptotic Bcl-2 genes are incorporated into an adenoviralinfectious particle to mediate gene transfer to a cell. Additionalexpression constructs encoding other therapeutic agents as describedherein may also be transferred via viral transduction using infectiousviral particles, for example, by transformation with an adenovirusvector of the present invention as described herein below.Alternatively, retroviral or bovine papilloma virus may be employed,both of which permit permanent transformation of a host cell with agene(s) of interest. Thus, in one example, viral infection of cells isused in order to deliver therapeutically significant genes to a cell.Typically, the virus simply will be exposed to the appropriate host cellunder physiologic conditions, permitting uptake of the virus. Thoughadenovirus is exemplified, the present methods may be advantageouslyemployed with other viral vectors, as discussed below.

Adenovirus. Adenovirus is particularly suitable for use as a genetransfer vector because of its mid-sized DNA genome, ease ofmanipulation, high titer, wide target-cell range, and high infectivity.The roughly 36 kB viral genome is bounded by 100-200 base pair (bp)inverted terminal repeats (ITR), in which are contained cis-actingelements necessary for viral DNA replication and packaging. The early(E) and late (L) regions of the genome that contain differenttranscription units are divided by the onset of viral DNA replication.

The E1 region (E1A and E1B) encodes proteins responsible for theregulation of transcription of the viral genome and a few cellulargenes. The expression of the E2 region (E2A and E2B) results in thesynthesis of the proteins for viral DNA replication. These proteins areinvolved in DNA replication, late gene expression, and host cell shutoff (Renan, 1990). The products of the late genes (L1, L2, L3, L4 andL5), including the majority of the viral capsid proteins, are expressedonly after significant processing of a single primary transcript issuedby the major late promoter (MLP). The MLP (located at 16.8 map units) isparticularly efficient during the late phase of infection, and all themRNAs issued from this promoter possess a 5′ tripartite leader (TL)sequence which makes them preferred mRNAs for translation.

In order for adenovirus to be optimized for gene therapy, it isnecessary to maximize the carrying capacity so that large segments ofDNA can be included. It also is very desirable to reduce the toxicityand immunologic reaction associated with certain adenoviral products.The two goals are, to an extent, coterminous in that elimination ofadenoviral genes serves both ends. By practice of the present invention,it is possible achieve both these goals while retaining the ability tomanipulate the therapeutic constructs with relative ease.

The large displacement of DNA is possible because the cis elementsrequired for viral DNA replication all are localized in the invertedterminal repeats (ITR) (100-200 bp) at either end of the linear viralgenome. Plasmids containing ITR's can replicate in the presence of anon-defective adenovirus (Hay et al., 1984). Therefore, inclusion ofthese elements in an adenoviral vector should permit replication.

In addition, the packaging signal for viral encapsidation is localizedbetween 194-385 bp (0.5-1.1 map units) at the left end of the viralgenome (Hearing et al., 1987). This signal mimics the proteinrecognition site in bacteriophage λ DNA where a specific sequence closeto the left end, but outside the cohesive end sequence, mediates thebinding to proteins that are required for insertion of the DNA into thehead structure. E1 substitution vectors of Ad have demonstrated that a450 bp (0-1.25 map units) fragment at the left end of the viral genomecould direct packaging in 293 cells (Levrero et al., 1991).

Previously, it has been shown that certain regions of the adenoviralgenome can be incorporated into the genome of mammalian cells and thegenes encoded thereby expressed. These cell lines are capable ofsupporting the replication of an adenoviral vector that is deficient inthe adenoviral function encoded by the cell line. There also have beenreports of complementation of replication deficient adenoviral vectorsby “helping” vectors, e.g., wild-type virus or conditionally defectivemutants.

Replication-deficient adenoviral vectors can be complemented, in trans,by helper virus. This observation alone does not permit isolation of thereplication-deficient vectors, however, since the presence of helpervirus, needed to provide replicative functions, would contaminate anypreparation. Thus, an additional element was needed that would addspecificity to the replication and/or packaging of thereplication-deficient vector. That element, as provided for in thepresent invention, derives from the packaging function of adenovirus.

It has been shown that a packaging signal for adenovirus exists in theleft end of the conventional adenovirus map (Tibbetts, 1977). Laterstudies showed that a mutant with a deletion in the E1A (194-358 bp)region of the genome grew poorly even in a cell line that complementedthe early (E1A) function (Hearing and Shenk, 1983). When a compensatingadenoviral DNA (0-353 bp) was recombined into the right end of themutant, the virus was packaged normally. Further mutational analysisidentified a short, repeated, position-dependent element in the left endof the Ad5 genome. One copy of the repeat was found to be sufficient forefficient packaging if present at either end of the genome, but not whenmoved towards the interior of the Ad5 DNA molecule (Hearing et al,1987).

By using mutated versions of the packaging signal, it is possible tocreate helper viruses that are packaged with varying efficiencies.Typically, the mutations are point mutations or deletions. When helperviruses with low efficiency packaging are grown in helper cells, thevirus is packaged, albeit at reduced rates compared to wild-type virus,thereby permitting propagation of the helper. When these helper virusesare grown in cells along with virus that contains wild-type packagingsignals, however, the wild-type packaging signals are recognizedpreferentially over the mutated versions. Given a limiting amount ofpackaging factor, the virus containing the wild-type signals arepackaged selectively when compared to the helpers. If the preference isgreat enough, stocks approaching homogeneity should be achieved.

Retrovirus. The retroviruses are a group of single-stranded RNA virusescharacterized by an ability to convert their RNA to double-stranded DNAin infected cells by a process of reverse-transcription (Coffin, 1990).The resulting DNA then stably integrates into cellular chromosomes as aprovirus and directs synthesis of viral proteins. The integrationresults in the retention of the viral gene sequences in the recipientcell and its descendants. The retroviral genome contains threegenes—gag, pol and env—that code for capsid proteins, polymerase enzyme,and envelope components, respectively. A sequence found upstream fromthe gag gene, termed Ψ, functions as a signal for packaging of thegenome into virions. Two long terminal repeat (LTR) sequences arepresent at the 5′ and 3′ ends of the viral genome. These contain strongpromoter and enhancer sequences and also are required for integration inthe host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding apromoter is inserted into the viral genome in the place of certain viralsequences to produce a virus that is replication-defective. In order toproduce virions, a packaging cell line containing the gag, pol and envgenes but without the LTR and Ψ components is constructed (Mann et al.,1983). When a recombinant plasmid containing a human cDNA, together withthe retroviral LTR and Ψ sequences is introduced into this cell line (bycalcium phosphate precipitation for example), the Ψ sequence allows theRNA transcript of the recombinant plasmid to be packaged into viralparticles, which are then secreted into the culture media (Nicolas andRubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containingthe recombinant retroviruses is collected, optionally concentrated, andused for gene transfer. Retroviral vectors are able to infect a broadvariety of cell types. However, integration and stable expression ofmany types of retroviruses require the division of host cells (Paskindet al., 1975).

An approach designed to allow specific targeting of retrovirus vectorsrecently was developed based on the chemical modification of aretrovirus by the chemical addition of galactose residues to the viralenvelope. This modification could permit the specific infection of cellssuch as hepatocytes via asialoglycoprotein receptors, should this bedesired.

A different approach to targeting of recombinant retroviruses wasdesigned in which biotinylated antibodies against a retroviral envelopeprotein and against a specific cell receptor were used. The antibodieswere coupled via the biotin components by using streptavidin (Roux etal., 1989). Using antibodies against major histocompatibility complexclass I and class II antigens, the infection of a variety of human cellsthat bore those surface antigens was demonstrated with an ecotropicvirus in vitro (Roux et al., 1989).

Adeno-associated Virus. AAV utilizes a linear, single-stranded DNA ofabout 4700 base pairs. Inverted terminal repeats flank the genome. Twogenes are present within the genome, giving rise to a number of distinctgene products. The first, the cap gene, produces three different virionproteins (VP), designated VP-1, VP-2 and VP-3. The second, the rep gene,encodes four non-structural proteins (NS). One or more of these rep geneproducts is responsible for transactivating AAV transcription.

The three promoters in AAV are designated by their location, in mapunits, in the genome. These are, from left to right, p5, p19 and p40.Transcription gives rise to six transcripts, two initiated at each ofthree promoters, with one of each pair being spliced. The splice site,derived from map units 42-46, is the same for each transcript. The fournon-structural proteins apparently are derived from the longer of thetranscripts, and three virion proteins all arise from the smallesttranscript.

AAV is not associated with any pathologic state in humans.Interestingly, for efficient replication, AAV requires “helping”functions from viruses such as herpes simplex virus I and II,cytomegalovirus, pseudorabies virus and, of course, adenovirus. The bestcharacterized of the helpers is adenovirus, and many “early” functionsfor this virus have been shown to assist with AAV replication. Low levelexpression of AAV rep proteins is believed to hold AAV structuralexpression in check, and helper virus infection is thought to removethis block.

The terminal repeats of the AAV vector can be obtained by restrictionendonuclease digestion of AAV or a plasmid such as p201, which containsa modified AAV genome (Samulski et al. 1987), or by other methods knownto the skilled artisan, including but not limited to chemical orenzymatic synthesis of the terminal repeats based upon the publishedsequence of AAV. The ordinarily skilled artisan can determine, bywell-known methods such as deletion analysis, the minimum sequence orpart of the AAV ITRs which is required to allow function, i.e., stableand site-specific integration. The ordinarily skilled artisan also candetermine which minor modifications of the sequence can be toleratedwhile maintaining the ability of the terminal repeats to direct stable,site-specific integration.

AAV-based vectors have proven to be safe and effective vehicles for genedelivery in vitro, and these vectors are being developed and tested inpre-clinical and clinical stages for a wide range of applications inpotential gene therapy, both ex vivo and in vivo (Carter and Flotte,1996; Chatterjee et al., 1995; Ferrari et al., 1996; Fisher et al.,1996; Flotte et al., 1993; Goodman et al., 1994; Kaplitt et al., 1994;1996, Kessler et al., 1996; Koeberl et al., 1997; Mizukami et al., 1996;Xiao et al., 1996).

AAV-mediated efficient gene transfer and expression in the lung has ledto clinical trials for the treatment of cystic fibrosis (Carter andFlotte, 1996; Flotte et al., 1993). Similarly, the prospects fortreatment of muscular dystrophy by AAV-mediated gene delivery of thedystrophin gene to skeletal muscle, of Parkinson's disease by tyrosinehydroxylase gene delivery to the brain, of hemophilia B by Factor 1×genedelivery to the liver, and potentially of myocardial infarction byvascular endothelial growth factor gene to the heart, appear promisingsince AAV-mediated transgene expression in these organs has recentlybeen shown to be highly efficient (Fisher et al., 1996; Flotte et al.,1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et al.,1996; Ping et al., 1996; Xiao et al., 1996).

Other Viral Vectors. Other viral vectors may be employed as expressionconstructs in the present invention. Vectors derived from viruses suchas vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar etal., 1988) canary pox virus, and herpes viruses may be employed. Theseviruses offer several features for use in gene transfer into variousmammalian cells.

ii. Non-Viral Transfer

DNA constructs of the present invention are generally delivered to acell, in certain situations, the nucleic acid to be transferred isnon-infectious, and can be transferred using non-viral methods.

Several non-viral methods for the transfer of expression constructs intocultured mammalian cells are contemplated by the present invention.These include calcium phosphate precipitation (Graham and Van Der Eb,1973; Chen and Okayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal,1985), electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984),direct microinjection (Harland and Weintraub, 1985), DNA-loadedliposomes (Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication(Fechheimer et al., 1987), gene bombardment using high velocitymicroprojectiles (Yang et al., 1990), and receptor-mediated transfection(Wu and Wu, 1987; Wu and Wu, 1988).

Once the construct has been delivered into the cell the nucleic acidencoding the therapeutic gene may be positioned and expressed atdifferent sites. In certain embodiments, the nucleic acid encoding thetherapeutic gene may be stably integrated into the genome of the cell.This integration may be in the cognate location and orientation viahomologous recombination (gene replacement) or it may be integrated in arandom, non-specific location (gene augmentation). In yet furtherembodiments, the nucleic acid may be stably maintained in the cell as aseparate, episomal segment of DNA. Such nucleic acid segments or“episomes” encode sequences sufficient to permit maintenance andreplication independent of or in synchronization with the host cellcycle. How the expression construct is delivered to a cell and where inthe cell the nucleic acid remains is dependent on the type of expressionconstruct employed.

In a particular embodiment of the invention, the expression constructmay be entrapped in a liposome. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh andBachhawat, 1991). The addition of DNA to cationic liposomes causes atopological transition from liposomes to optically birefringentliquid-crystalline condensed globules (Radler et al., 1997). TheseDNA-lipid complexes are potential non-viral vectors for use in genetherapy.

Liposome-mediated nucleic acid delivery and expression of foreign DNA invitro has been very successful. Using the β-lactamase gene, Wong et al.(1980) demonstrated the feasibility of liposome-mediated delivery andexpression of foreign DNA in cultured chick embryo, HeLa, and hepatomacells. Nicolau et al. (1987) accomplished successful liposome-mediatedgene transfer in rats after intravenous injection. Also included arevarious commercial approaches involving “lipofection” technology.

In certain embodiments of the invention, the liposome may be complexedwith a hemagglutinating virus (HVJ). This has been shown to facilitatefusion with the cell membrane and promote cell entry ofliposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments,the liposome may be complexed or employed in conjunction with nuclearnonhistone chromosomal proteins (HMG-1) (Kato et al., 1991). In yetfurther embodiments, the liposome may be complexed or employed inconjunction with both HVJ and HMG-1. In that such expression constructshave been successfully employed in transfer and expression of nucleicacid in vitro and in vivo, then they are applicable for the presentinvention.

Other vector delivery systems which can be employed to deliver a nucleicacid encoding a therapeutic gene into cells are receptor-mediateddelivery vehicles. These take advantage of the selective uptake ofmacromolecules by receptor-mediated endocytosis in almost all eukaryoticcells. Because of the cell type-specific distribution of variousreceptors, the delivery can be highly specific (Wu and Wu, 1993).

Receptor-mediated gene targeting vehicles generally consist of twocomponents: a cell receptor-specific ligand and a DNA-binding agent.Several ligands have been used for receptor-mediated gene transfer. Themost extensively characterized ligands are asialoorosomucoid (ASOR) (Wuand Wu, 1987) and transferring (Wagner et al., 1990). Recently, asynthetic neoglycoprotein, which recognizes the same receptor as ASOR,has been used as a gene delivery vehicle (Ferkol et al., 1993; Peraleset al., 1994) and epidermal growth factor (EGF) has also been used todeliver genes to squamous carcinoma cells (Myers, EPO 0273085).

In other embodiments, the delivery vehicle may comprise a ligand and aliposome. For example, Nicolau et al. (1987) employed lactosyl-ceramide,a galactose-terminal asialganglioside, incorporated into liposomes andobserved an increase in the uptake of the insulin gene by hepatocytes.Thus, it is feasible that a nucleic acid encoding a therapeutic genealso may be specifically delivered into a cell type such as prostate,epithelial or tumor cells, by any number of receptor-ligand systems withor without liposomes. For example, the human prostate-specific antigen(Watt et al., 1986) may be used as the receptor for mediated delivery ofa nucleic acid in prostate tissue.

In another embodiment of the invention, the expression construct maysimply consist of naked recombinant DNA or plasmids. Transfer of theconstruct may be performed by any of the methods mentioned above whichphysically or chemically permeabilize the cell membrane. This isapplicable particularly for transfer in vitro, however, it may beapplied for in vivo use as well. Dubensky et al. (1984) successfullyinjected polyomavirus DNA in the form of CaPO₄ precipitates into liverand spleen of adult and newborn mice demonstrating active viralreplication and acute infection. Benvenisty and Neshif (1986) alsodemonstrated that direct intraperitoneal injection of CaPO₄ precipitatedplasmids results in expression of the transfected genes. It isenvisioned that DNA encoding a CAM may also be transferred in a similarmanner in vivo and express CAM.

Another embodiment of the invention for transferring a naked DNAexpression construct into cells may involve particle bombardment. Thismethod depends on the ability to accelerate DNA coated microprojectilesto a high velocity allowing them to pierce cell membranes and entercells without killing them (Klein et al., 1987). Several devices foraccelerating small particles have been developed. One such device relieson a high voltage discharge to generate an electrical current, which inturn provides the motive force (Yang et al., 1990). The microprojectilesused have consisted of biologically inert substances such as tungsten orgold beads

F. Gene Delivery System for Toxic Gene Products

It is now known that programmed cell death, or apoptosis, plays animportant role in development, homeostasis, and disease processes. It iscontemplated in the present invention, that genes involved in apoptoticpathways may be useful in the treatment of diseases related to disordersin these pathways. In another embodiment, the use of non-pro-apoptic,cytotoxic genes are contemplated for use in treating hyperproliferativeand other disease states in which cell death would be therapeutic.

The use of proapoptotic genes to treat cancers was proposed severalyears ago (Fisher, 1994; Thompson, 1995). However, the expression ofpro-apoptic genes often results in death of the host cell if theirexpression is not regulated. In another embodiment of the presentinvention, it is contemplated that a novel co-transfer vector system isused to permit delivery of vectors that express potentially toxic genes.For example, the expression of Bcl-2 family member via gene transfer maybe valuable in the treatment of a variety of hyperproliferativediseases, such as cancer. However, constructing an adenoviral vectorexpressing a pro-apoptic gene driven by a constitutive promoter becomesproblematic in the packaging cell, presumably because of its highapoptotic activity (i.e., cell toxicity).

In one embodiment, the inventors demonstrate a system for safelyinducing the expression of the bax gene in a host cell byadenovirus-mediated gene co-transfer. The system provides a firstadenoviral vector containing a human gene wherein the expression productis cytotoxic. The cytotoxic gene is driven by a promoter, not active inthe host or target cell. A second adenoviral vector is provided, whereinthe gene, under the control of a promoter, encodes a transactivatingprotein. Induction of the promoter driving the expression of thetransactivating protein, initiates the expression of the cytotoxic geneproduct.

Experimental data demonstrate that the vector expresses a minimalbackground level of bax protein in cultured mammalian cells thuspreventing apoptosis of packaging cells. The expression of the bax genewas substantially induced both in vitro and in vivo by transferring itinto target cells along with of an adenoviral vector expressing thetransactivator, fusion protein GAL4/VP16. Thus, adenovirus-mediated geneco-transfer permits the regulated expression of bax via the inducibleexpression of the GAL4/VP16 gene. In other embodiments of the invention,the pro-apoptic genes Bak, Bim, Bik, Bid, Bad and Harakiri arecontemplated for use in adenovirus-mediated gene co-transfer

Adenovirus-mediated gene co-transfer is not limited to proapoptoticgenes or a specific promoter. It also is contemplated that co-transfersystem could be used to treat various hyperproliferative diseases,wherein regulating the expression of a toxic gene product is desired.Depending on the tissue being treated, a tissue specific promoter couldbe chosen to permit in vivo transactivation only in the target tissue.For example, co-transfer of a tumor suppressor gene linked to a promoterand a vector expressing a transactivator that specifically binds to thepromoter, would be useful in treating hyperproliferative diseases. Thus,in one embodiment of the invention, a promoter linked to a particulargene can be selected to provide tissue specific expression. Regulatedco-transfer expression of other toxic gene products also arecontemplated and discussed below.

i. Vector Co-Transfer and Promoters

The use of proapoptotic genes to treat cancers via gene therapy has notbeen reported, possibly due to the difficulty in constructing vectorsthat can efficiently transduce target cells with such genes. Forexample, Larregina et al. showed that constructing an adenovirusexpressing the Fas-Ligand (Fas-L) was difficult because Fas-L inducesapoptosis in 293 packaging cells, (Larregina et al., 1998). Arai et al.achieved efficient antitumor activity by adenovirus-mediated Fas-L genetransfer, but this required the use of 293 cells resistant to Fas-L orcaspase inhibitor for vector production, (Arai et al., 1997).

The gene co-transfer system of the present invention overcomes theseobstacles, by providing a pro-apoptic gene linked to a regulatable,promoter. The regulatable promoter prevents expression of thepro-apoptic gene in the host or packaging cell, which would result incell death. The expression of the pro-apoptic gene is induced by atransactivator protein, carried by a gene on a second expression vector.In one embodiment of the present invention, adenovirus-mediated geneco-transfer uses a first adenovirus comprising human bax cDNA driven bya promoter consisting of a heptamer of GAL4-binding sites and a TATAbox. A second adenovirus (i.e., co-transfer) comprising the GAL4/VP16transactivator fusion protein linked to a regulatable promoter operablein eukaryotic cells, is provided to selectively induce bax expression.It is contemplated in other embodiments, that the first promoter can bethe ecdysone-responsive promoter or Tet-On™ and the inducer of the firstpromoter ecdysone or muristeron A and doxycycline, respectively. For acomplete description of the ecdysone system and Tet-On™ see section D,herein. It also is contemplated, that that the first promoter can be theHIV-1 LTR or HIV-2 LTR and the inducer of the first promoter tat. It iscontemplated in other embodiments, that yeast, E. coli and insectpromoters may also be useful in the present invention for regulatedexpression of cytotoxic genes.

For human or mammalian cytotoxic gene therapy via vector-mediated geneco-transfer, the Bcl-2 genes Bak, Bim, Bik, Bid, Bad and Harakiri arecontemplated as useful in the present invention. Additional cytotoxicgene products contemplated as useful in the present invention, aredescribed below.

It is contemplated in the present invention that a gene encoding atransactivating protein is supplied by a second vector. In certainembodiments, the gene encoding the transactivating protein can be linkedto a constitutive promoter. In other embodiments, the gene encoding thetransactivating protein can be under the control of an induciblepromoter, permitting regulated expression of the transactivatingprotein. Pancreatic, liver, skeletal muscle, smooth muscle, skin, lung,endothelium and blood are some examples of tissues in which tissuespecific promoters might be selected for use. Table 2, Table 3 and Table4 provide a list of some useful tissue specific promoters,promoter/enhancers and inducible promoter/enhancers, respectively, thatmay be used in combination and are considered useful in the presentinvention.

An important consideration when selecting a promoter to drive theexpression of the cytotoxic gene product on the first vector, is thatthe transactivating protein (i.e., inducer) is not active in the hostcell. For example, if the host cell expresses a transactivating protein,capable of activating the promoter on the first expression vector,upregulation of the cytotoxic gene may result.

The vector-mediated co-transfer system is particularly useful in vivo.In such embodiments, it may be desirable that transactivating proteinalso is not active in the target cell. The presence of thetransactivating protein in the target cell would limit the temporal useof the co-delivery system, as the transactivating protein would bepresent at the time of delivery of the cytotoxic expression construct.

A variety of transactivating genes theoretically can be chosen toexpress transactivating protein factors, to drive the expression of atoxic gene on the first vector. Another consideration in choosing atransactivating protein factor is its efficacy of transcriptionalactivation in a given tissue type. It may be that a particular tissuespecific transactivating factor has low levels of cross tissue activity,which could potentially be cytotoxic to healthy, normal cell or tissuetypes.

ii. In Vitro and In Vivo Delivery of Vectors to Target Cells

An adenoviral vector expressing a Blc-2 member gene, would facilitatethe therapeutic evaluation of the Bcl-2 member gene, since such a vectorwould have potentially high transduction efficiencies in a variety oftissues. However, constructing an adenoviral vector that can express baxfor example, has been problematic, presumably because of the bax gene'shigh apoptotic activity and its toxic effect on packaging 293 cells(Rosse et al., 1998). It is contemplated that vector-mediated geneco-transfer of the present invention will be useful for regulating bothin vitro and in vivo expression of potentially cytotoxic gene products

In one embodiment of the present invention, the in vitro expression oftherapeutic genes are considered. In one example, shuttle plasmids inwhich bax cDNA was driven by a GAL4-responsive promoter consisting offive GAL4-binding sites and a TATA box (GT) were constructed.Recombinant viral vectors (Ad) were obtained after a single in vitrotransfection of 293 cells with pAd/GT-Bax plus a 35-kb ClaI fragmentfrom Ad/p53, (Zhang et al., 1993). Virus from a single plaque wasexpanded in 293 cells, twice purified and vector titer determined to be3.3×10¹² viral particles/ml. Thus, the vector-mediated gene co-transfersystem allows the in vitro replication of Ad/GT-Bax particles (3.3×10¹²viral particles/ml) in the host cell (e.g., 293 cells), without killingthe host cell (i.e. no Bax expression). The functionality of Ad/GT-Baxin vitro was documented by the co-transfer of Ad/GT-Bax and thetransactivator Ad/PGK-GV16 to the cultured human lung carcinoma cellline H1299, demonstrating the induction of Bax expression viaco-transfer. The in vitro expression of Bax in the vector-mediated geneco-transfer was also demonstrated to promote apoptosis in human lungcancer cell lines.

In other embodiments, the induction of therapeutic gene expression invivo is contemplated for use in the present invention. Thus, in anotherexample, to test whether bax gene expression could be similarly inducedby adenovirus-mediated gene codelivery in vivo, adult Balb/c mice wereinfused via their tail veins with Ad/GT-Bax plus Ad/PGK-GV16, at a totalvector dose of 6×10¹⁰ particles/mouse and a vector ratio of 2:1. Micewere then sacrificed at 24 h after treatment, after which liver sampleswere harvested for western blot analysis and histopathologicalexamination. A 14-fold increase in bax protein levels in animals treatedwith Ad/GT-Bax plus Ad/PGK-GV16 relative to control animals was observedas well as apoptosis of normal liver cells. These results demonstratethat bax expression can regulated in vivo by expressing GAL4/VP16protein via the adenovirus-mediated gene co-transfer system.

In certain embodiments of the invention, the temporal sequence ofvector-mediated co-transfer delivery is contemplated. In one embodiment,the vectors are delivered simultaneously. In other embodiments, theexpression vector encoding the cytotoxic gene is delivered first,followed by the expression vector encoding the transactivating protein.In still other embodiments, the expression vector encoding thetransactivating protein is delivered first, followed by the expressionvector encoding the cytotoxic gene. The time between delivery of thefirst vector and the second vector is dependent on various parameters.Parameters to be considered when formulating a protocol include, but arenot limited to, vector transducing efficiency, transducing cell type,efficiency of cytotoxic gene expression, efficiency of transactivatinggene expression, cytotoxic protein stability and transactivating proteinstability.

iii. Viral and Non-Viral Vectors

It is contemplated in the present invention, that gene co-transfer canbe employed using any vector (i.e., viral, plasmid, shuttle vector). Thetherapeutic gene as described above, can be incorporated into anadenoviral infectious particle to mediate gene transfer to a cell.Alternatively, retrovirus, adeno-associated virus, vaccinia virus,canary pox virus, herpes virus, canary pox virus and reovirus also arecontemplated as gene transfer vectors for use in the present invention.

In certain embodiments, non-viral vectors, such as plasmids, shuttleplasmids and cosmids are contemplated for use. Non-viral methods for thetransfer of expression constructs into cultured mammalian cells includecalcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen andOkayama, 1987; Rippe et al., 1990) DEAE-dextran (Gopal, 1985),electroporation (Tur-Kaspa et al., 1986; Potter et al., 1984), directmicroinjection (Harland and Weintraub, 1985), DNA-loaded liposomes(Nicolau and Sene, 1982; Fraley et al., 1979), cell sonication(Fechheimer et al., 1987), gene bombardment using high velocitymicroprojectiles (Yang et al., 1990), and receptor-mediated transfection(Wu and Wu, 1987; Wu and Wu, 1988). For a more detailed description ofboth viral and non-viral methods and applications of gene transfer,refer to section F.

iv. Other Genes Toxic to Host Cells

In other embodiments of the present invention, the use of geneco-transfer system is contemplated for use in delivering non-pro-apoptictherapeutic genes that express potentially cytotoxic gene products. Itis contemplated, that cancer, hyperproliferative (e.g., psoriasis,cytys) and inflammatory conditions (e.g. rheumatoid arthritis,allergies) could be treated by using the gene co-transfer system, bytargeting these cells with genes that encode potentially cytotoxicproducts. It is contemplated that genes encoding cytokines (e.g.,interferons), toxins antisense constructs, ribozymes, single chainantibodies, proteases and antigens would be useful in particulartherapies, and that the co-transfer method will allow regulatedexpression of these genes.

In certain embodiments, various toxins are contemplated to be useful aspart of the expression vectors of the present invention, these toxinsinclude bacterial toxins such as ricin A-chain (Burbage, 1997),diphtheria toxin A (Massuda et al., 1997; Lidor, 1997), pertussis toxinA subunit, E. coli enterotoxin toxin A subunit, cholera toxin A subunitand pseudomonas toxin c-terminal. Recently, it was demonstrated thattransfection of a plasmid containing the fusion protein regulatablediphtheria toxin A chain gene was cytotoxic for cancer cells. Thus, genetransfer of regulated toxin genes might also be applied to the treatmentof cancers or other hyperproliferative diseases (Massuda et al., 1997).

In certain embodiments, cytokines such as IL-1, IL-2, IL-3, IL-4, IL-5,IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF, oncostatin M, TGF-β,TNF-α, TNF-β and G-CSF are contemplated for use in the vector-mediatedco-transfer system.

In other embodiments, antisense constructs are contemplated for use inthe present invention. Antisense methodology takes advantage of the factthat nucleic acids tend to pair with “complementary” sequences.Antisense polynucleotides, when introduced into a target cell,specifically bind to their target polynucleotide and interfere withtranscription, RNA processing, transport, translation and/or stability.Antisense RNA constructs, or DNA encoding such antisense RNA's, may beemployed to inhibit gene transcription or translation or both within ahost cell, either in vitro or in vivo, such as within a host animal,including a human subject. Engineering antisense constructs is coveredin detail in Section D. Particular oncogenes that are targets forantisense constructs are ras, myc, neu, raf erb, src, fms, jun, trk,ret, hst, gsp and abl. Also contemplated to be useful will beanti-apoptotic genes such as Bcl-2, Mcl-1, A1 and Bfl-1

In still other embodiments, ribozymes are considered for use in thepresent invention. Although proteins traditionally have been used forcatalysis of nucleic acids, another class of macromolecules has emergedas useful in this endeavor. Ribozymes are RNA-protein complexes thatcleave nucleic acids in a site-specific fashion. Ribozymes have specificcatalytic domains that possess endonuclease activity (Kim and Cook,1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, alarge number of ribozymes accelerate phosphoester transfer reactionswith a high degree of specificity, often cleaving only one of severalphosphoesters in an oligonucleotide substrate (Cook et al., 1981; Micheland Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity hasbeen attributed to the requirement that the substrate bind via specificbase-pairing interactions to the internal guide sequence (“IGS”) of theribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part ofsequence-specific cleavage/ligation reactions involving nucleic acids(Joyce, 1989; Cook et al., 1981). For example, U.S. Pat. No. 5,354,855reports that certain ribozymes can act as endonucleases with a sequencespecificity greater than that of known ribonucleases and approachingthat of the DNA restriction enzymes. Thus, sequence-specificribozyme-mediated inhibition of gene expression may be particularlysuited to therapeutic applications (Scanlon et al., 1991; Sarver et al.,1990). Recently, it was reported that ribozymes elicited genetic changesin some cells lines to which they were applied; the altered genesincluded the oncogenes H-ras, c-fos and genes of HIV. Most of this workinvolved the modification of a target mRNA, based on a specific mutantcodon that is cleaved by a specific ribozyme. Targets for thisembodiment will include oncogenes such as ras, myc, neu, raf, erb, src,fins, jun, trk, ret, hst, gsp, bcl-2, EGFR, grb2 and abl. Otherconstructs will include overexpression of antiapoptotic genes such asbcl-2.

In yet another embodiment, one gene may comprise a single-chainantibody. Methods for the production of single-chain antibodies are wellknown to those of skill in the art. The skilled artisan is referred toU.S. Pat. No. 5,359,046, (incorporated herein by reference) for suchmethods and section D above.

Antibodies to a wide variety of molecules are contemplated, such asoncogenes, growth factors, hormones, enzymes, transcription factors orreceptors.

In certain embodiments, it may be useful to express enzymes, that arepotentially cytotoxic. For example, the protease caspase-7, has beenimplicated in apoptosis and thus potentially useful in gene therapy(Marcelli et al., 1999). One could express a variety of proteases, whichhave either been genetically engineered to function at physiological pHand/or active without enzymatic processing (Briand et al., 1999).Alternatively, proteases can be cloned from thermostable or pH stableorganisms (Choi et al., 1999; Sundd et al., 1998). Thus, one couldexpress a protease in a given cell and potentially inactivate viaproteolysis, key metabolic and signaling proteins, needed for cellviability.

In another embodiment, treatment of protein folding disorders via thegene co-transfer system are contemplated. For example, Cruetzfeldt-Jakobdisease, Kuru, the human transmissible bovine spongiform encephalopathy(e.g., mad cow disease) and scrappie in sheep, are diseases related tocellular prion protein misfolding (Grandien and Wahren, 1998; Buschmannet al., 1998; Hill et al., 1999) The disease state ensues when anindividual is exposesed to an infectious (mutated) form of the prionprotein. This infectious prion protein (PrP(Sc)) acts as a misfoldingcatalyst or scaffold, and induces conformational changes in anindividuals native prion proteins (PrP(C)), leading to the intraneuronalaccumulation of a pathological prion isoform. Prions replicate inlymphoreticular tissues before neuroinvasion and have been demonstratedto be detectable via tonsil biopsy (Hill et al., 1999). It might bepossible using vector-mediated co-transfer, to provide antisense mRNA topatients who test positive for PrP(Sc), to prevent transcription ofprion mRNA and thus block protein synthesis. Alternatively, expressionof cytokines could be targeted to lymphoreticular tissues, expression ofproteases or specific antigens could be used to tag these cells fordestruction, reducing prion protein expression. It is contemplatedfurther in the present invention, that Alzheimer's disease could betreated similarly using vector-mediated co-transfer

G. Pharmaceuticals and Methods of Treating Cancer

In a particular aspect, the present invention provides methods for thetreatment of various malignancies. Treatment methods will involvetreating an individual with an effective amount of a viral particle, asdescribed above, containing a therapeutic gene of interest. An effectiveamount is described, generally, as that amount sufficient to detectablyand repeatedly to ameliorate, reduce, minimize or limit the extent of adisease or its symptoms. More rigorous definitions may apply, includingelimination, eradication or cure of disease.

To kill cells, inhibit cell growth, inhibit metastasis, decrease tumorsize and otherwise reverse or reduce the malignant phenotype of tumorcells, using the methods and compositions of the present invention, onewould generally contact a “target” cell with the therapeutic expressionconstruct. This may be combined with compositions comprising otheragents effective in the treatment of cancer. These compositions would beprovided in a combined amount effective to kill or inhibit proliferationof the cell. This process may involve contacting the cells with theexpression construct and the agent(s) or factor(s) at the same time.This may be achieved by contacting the cell with a single composition orpharmacological formulation that includes both agents, or by contactingthe cell with two distinct compositions or formulations, at the sametime, wherein one composition includes the expression construct and theother includes the second agent.

Alternatively, the gene therapy may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent and expression construct are applied separately tothe cell, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the agentand expression construct would still be able to exert an advantageouslycombined effect on the cell. In such instances, it is contemplated thatone would contact the cell with both modalities within about 12-24 h ofeach other and, more preferably, within about 6-12 h of each other, witha delay time of only about 12 h being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several d (2, 3, 4, 5, 6 or 7) to severalwk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

Administration of the therapeutic expression constructs of the presentinvention to a patient will follow general protocols for theadministration of chemotherapeutics, taking into account the toxicity,if any, of the vector. It is expected that the treatment cycles would berepeated as necessary. It also is contemplated that various standardtherapies, as well as surgical intervention, may be applied incombination with the described gene therapy.

Where clinical application of a gene therapy is contemplated, it will benecessary to prepare the complex as a pharmaceutical compositionappropriate for the intended application. Generally this will entailpreparing a pharmaceutical composition that is essentially free ofpyrogens, as well as any other impurities that could be harmful tohumans or animals. One also will generally desire to employ appropriatesalts and buffers to render the complex stable and allow for complexuptake by target cells.

Aqueous compositions of the present invention comprise an effectiveamount of the compound, dissolved or dispersed in a pharmaceuticallyacceptable carrier or aqueous medium. Such compositions can also bereferred to as inocula. The phrases “pharmaceutically orpharmacologically acceptable” refer to molecular entities andcompositions that do not produce an adverse, allergic or other untowardreaction when administered to an animal, or a human, as appropriate. Asused herein, “pharmaceutically acceptable carrier” includes any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients also canbe incorporated into the compositions.

The compositions of the present invention may include classicpharmaceutical preparations. Dispersions also can be prepared inglycerol, liquid polyethylene glycols, and mixtures thereof and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms.

Depending on the particular cancer to be, administration of therapeuticcompositions according to the present invention will be via any commonroute so long as the target tissue is available via that route. Thisincludes oral, nasal, buccal, rectal, vaginal or topical. Topicaladministration would be particularly advantageous for treatment of skincancers. Alternatively, administration will be by orthotopic,intradermal, subcutaneous, intramuscular, intraperitoneal or intravenousinjection. Such compositions would normally be administered aspharmaceutically acceptable compositions that include physiologicallyacceptable carriers, buffers or other excipients.

In certain embodiments, ex vivo therapies also are contemplated. Ex vivotherapies involve the removal, from a patient, of target cells. Thecells are treated outside the patient's body and then returned. Oneexample of ex vivo therapy would involve a variation of autologous bonemarrow transplant. Many times, ABMT fails because some cancer cells arepresent in the withdrawn bone marrow, and return of the bone marrow tothe treated patient results in repopulation of the patient with cancercells. In one embodiment, however, the withdrawn bone marrow cells couldbe treated while outside the patient with an viral particle that targetsand kills the cancer cell. Once the bone marrow cells are “purged,” theycan be reintroduced into the patient.

The treatments may include various “unit doses.” Unit dose is defined ascontaining a predetermined-quantity of the therapeutic compositioncalculated to produce the desired responses in association with itsadministration, i.e., the appropriate route and treatment regimen. Thequantity to be administered, and the particular route and formulation,are within the skill of those in the clinical arts. Also of import isthe subject to be treated, in particular, the state of the subject andthe protection desired. A unit dose need not be administered as a singleinjection but may comprise continuous infusion over a set period oftime. Unit dose of the present invention may conveniently may bedescribed in terms of plaque forming units (pfu) of the viral construct.Unit doses range from 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹,10¹², 10¹³ pfu and higher.

Preferably, patients will have adequate bone marrow function (defined asa peripheral absolute granulocyte count of >2,000/mm³ and a plateletcount of 100,000/mm³), adequate liver function (bilirubin <1.5 mg/dl)and adequate renal function (creatinine <1.5 mg/dl).

i) Cancer Therapy

One of the preferred embodiments of the present invention involves theuse of viral vectors to deliver therapeutic genes to cancer cells.Target cancer cells include cancers of the lung, brain, prostate,kidney, liver, ovary, breast, skin, stomach, esophagus, head and neck,testicles, colon, cervix, lymphatic system and blood. Of particularinterest are non-small cell lung carcinomas including squamous cellcarcinomas, adenocarcinomas and large cell undifferentiated carcinomas.

According to the present invention, one may treat the cancer by directlyinjection a tumor with the viral vector. Alternatively, the tumor may beinfused or perfused with the vector using any suitable delivery vehicle.Local or regional administration, with respect to the tumor, also iscontemplated. Finally, systemic administration may be performed.Continuous administration also may be applied where appropriate, forexample, where a tumor is excised and the tumor bed is treated toeliminate residual, microscopic disease. Delivery via syringe orcatherization is preferred. Such continuous perfusion may take place fora period from about 1-2 hours, to about 2-6 hours, to about 6-12 hours,to about 12-24 hours, to about 1-2 days, to about 1-2 wk or longerfollowing the initiation of treatment. Generally, the dose of thetherapeutic composition via continuous perfusion will be equivalent tothat given by a single or multiple injections, adjusted over a period oftime during which the perfusion occurs.

For tumors of >4 cm, the volume to be administered will be about 4-10 ml(preferably 10 ml), while for tumors of <4 cm, a volume of about 1-3 mlwill be used (preferably 3 ml). Multiple injections delivered as singledose comprise about 0.1 to about 0.5 ml volumes. The viral particles mayadvantageously be contacted by administering multiple injections to thetumor, spaced at approximately 1 cm intervals.

In certain embodiments, the tumor being treated may not, at leastinitially, be resectable. Treatments with therapeutic viral constructsmay increase the resectability of the tumor due to shrinkage at themargins or by elimination of certain particularly invasive portions.Following treatments, resection may be possible. Additional viraltreatments subsequent to resection will serve to eliminate microscopicresidual disease at the tumor site.

A typical course of treatment, for a primary tumor or a post-excisiontumor bed, will involve multiple doses. Typical primary tumor treatmentinvolves a 6 dose application over a two-week period. The two-weekregimen may be repeated one, two, three, four, five, six or more times.During a course of treatment, the need to complete the planned dosingsmay be re-evaluated.

Cancer therapies also include a variety of combination therapies withboth chemical and radiation based treatments. Combination chemotherapiesinclude, for example, cisplatin (CDDP), carboplatin, procarbazine,mechlorethamine, cyclophosphamide, camptothecin, ifosfamide, melphalan,chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin,doxorubicin, bleomycin, plicomycin, mitomycin, etoposide (VP16),tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastinand methotrexate or any analog or derivative variant thereof.

Other factors that cause DNA damage and have been used extensivelyinclude what are commonly known as γ-rays, X-rays, and/or the directeddelivery of radioisotopes to tumor cells. Other forms of DNA damagingfactors are also contemplated such as microwaves and UV-irradiation. Itis most likely that all of these factors effect a broad range of damageon DNA, on the precursors of DNA, on the replication and repair of DNA,and on the assembly and maintenance of chromosomes. Dosage ranges forX-rays range from daily doses of 50 to 200 roentgens for prolongedperiods of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.Dosage ranges for radioisotopes vary widely, and depend on the half-lifeof the isotope, the strength and type of radiation emitted, and theuptake by the neoplastic cells.

Various combinations may be employed, gene therapy is “A” and the radio-or chemotherapeutic agent is “B”: A/B/A B/A/B B/B/A A/A/B A/B/B B/A/AA/B/B/B B/A/B/B B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/AB/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

The terms “contacted” and “exposed,” when applied to a cell, are usedherein to describe the process by which a therapeutic construct and achemotherapeutic or radiotherapeutic agent are delivered to a targetcell or are placed in direct juxtaposition with the target cell. Toachieve cell killing or stasis, both agents are delivered to a cell in acombined amount effective to kill the cell or prevent it from dividing.

The therapeutic compositions of the present invention are advantageouslyadministered in the form of injectable compositions either as liquidsolutions or suspensions; solid forms suitable for solution in, orsuspension in, liquid prior to injection may also be prepared. Thesepreparations also may be emulsified. A typical composition for suchpurpose comprises a pharmaceutically acceptable carrier. For instance,the composition may contain 10 mg, 25 mg, 50 mg or up to about 100 mg ofhuman serum albumin per milliliter of phosphate buffered saline. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oil and injectable organic esters such asethyloleate. Aqueous carriers include water, alcoholic/aqueoussolutions, saline solutions, parenteral vehicles such as sodiumchloride, Ringer's dextrose, etc. Intravenous vehicles include fluid andnutrient replenishers. Preservatives include antimicrobial agents,anti-oxidants, chelating agents and inert gases. The pH and exactconcentration of the various components the pharmaceutical compositionare adjusted according to well known parameters.

Additional formulations are suitable for oral administration. Oralformulations include such typical excipients as, for example,pharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate and the like. Thecompositions take the form of solutions, suspensions, tablets, pills,capsules, sustained release formulations or powders. When the route istopical, the form may be a cream, ointment, salve or spray.

H. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Apoptotic Mechanisms Following Adenovirus-Mediated p53Replacement Gene Therapy

Induction of program cell death pathway is a critical step in mostanticancer therapies including adenovirus mediated wild-type p53 genetherapy. The transient expression of the adenovirus vector requireseither induction of apoptosis, terminal differentiation, or cellularsenescence in order to result in effective therapy. As the a furtherunderstanding of the mechanisms involved in this process is gained, thiswill enable us to design more effective therapeutic approaches toanticancer treatment.

Materials and Methods

Cell Culture. H358 and H1299 are non-small cell lung cancer cell lineswith both copies of the p53 deleted and were obtained from A. Gazdar andJ. Minna. H322j is a non-small cell lung cancer cell line with a p53mutation. Cells were maintained in RPMI-1640 medium supplemented with10% fetal calf serum, 10 mM glutamine, 100 units/ml of penicillin, 100μg/ml of streptomycin, and 0.25 μg/ml of amphotericin B (Gibco-BRL, LifeTechnologies, Inc., Grand Island, N.Y.) and incubated at 37° C. in a 5%CO₂ incubator.

Adenovirus production. The construction and properties of the Adp53 havebeen reported elsewhere (Fujiwara et al., 1994; Zhang et al., 1993). TheAd5/CMV/β-gal virus was obtained from F. Graham, McMaster University,Hamilton, Ontario. The E1 deleted vector dl312 (obtained from T. Shenk,Princeton, N.J.) was utilized as a control vector. Adenovirus wasprepared as previously described (Graham and Prevec, 1991) and purifiedby two rounds of cesium chloride ultracentrifugation. Purified virus wasmixed with 10% glycerol and dialyzed twice against 1000 ml of a buffercontaining 10 mM Tris HCl (pH 7.5), 1/μM MgCl₂, and 10% glycerol at 4°C. for 6 h. Purified virus was aliquoted and stored at −80° C. untilused. Viral titer was determined by UV-spectrophotometric analysis(viral particles/ml) and by plaque assay (pfu/ml) (Zhang et al., 1995).Final viral concentrations for in vitro and in vivo infections were madeby dilution of stock virus in PBS. Adenovirus preparations were free ofreplication-competent adenovirus as determined by previously describedtechniques (Zhang et al., 1995).

Gene delivery. In vitro transfection studies for all cell lines wereperformed by plating 5×10⁵ cells in 100 mm plates (Falcon Plastics,Lincoln Park, N.J.). Forty-eight h after plating, cells were incubatedfor 2 h with purified virus in 2 mls of RPMI-1640 medium supplementedwith 2% fetal calf serum. The multiplicity of infection (MOI) was basedon cell counts of untreated plates. The MOI used for each cell line waschosen to result in an approximately 70-80% transduction based onpreliminary studies using the Ad5/CMV/β-gal vector. These were an MOI of5 pfu for the H1299 cell line, 70 pfu for the H358 cell line and 50 pfufor the H322j cell line. After 2 h, fresh RPMI-1640 medium supplementedwith 10% fetal calf serum was added to the plates. Cells and cell lysatewere collected at 6 h intervals up to 36 h following infection forwestern blot, cell cycle, and TUNEL assay analysis. This time course waschosen based on preliminary data indicating a large fraction ofapoptotic cells were evident at these times and later time pointsresulted in the observance of degraded cellular proteins.

Western blot analysis. Total cell lysates were prepared by lysingmonolayered cells in dishes with sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE) sample buffer after rinsing cells withphosphate buffered saline (PBS). Each lane was loaded with 50 μg of celllysate protein as determined by BCA protein assay (Pierce, Rockford,Ill.). After SDS PAGE at 100 volts for two h, the proteins in the gelswere transferred to hybond-ECL membrane (Amersham International PLC,Little Chalfont, Buckingham Shire, England). Membranes were blocked with3% milk and 0.1% Tween 20 (Sigma Chemical Company) in PBS and incubatedwith antibody against the specified protein overnight at 4° C. The mouseanti-human p53 (D0-7) (Pharmigen, San Diego, Calif.), mouse anti-humanBcl-2 (124) (Dako Corp., Carpintenia, Calif.), mouse anti-human Bak(Oncogene Science), mouse anti-human Bax (Pharmigen, San Diego, Calif.),mouse anti-human Bcl-x_(L) (Pharmigen, San Diego, Calif.), and mouseanti-human (β-actin monoclonal antibody (N350) (Amersham InternationalPLC, Buckingham Shire, England) were used. The membranes were developedaccording to Amershams ECL western blotting protocol.

Flow cytometry analysis for cell cycle. To measure the DNA histogram,cells were fixed in 70% ethanol at 4° C. for greater than 24 h. Thecells were incubated in propidium iodide (20 μg/ml) and ribonucleases(20 μg/ml) for 30 min at 37° C. All measures were made with an EpicsProfile II (Coulter Corp., Hialeath, Fla.) equipped with an air-cooledargon ion laser admitting 488 NM at 15 MW. A minimum of 10,000 eventsper sample were analyzed and FITC fluorescence was collected using a 525BP filter. Coulters cytologic program was used for data analysis. Meanpeak fluorescence was determined for each histogram.

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick endlabeling (TUNEL) Assay. The TUNEL assay was performed utilizing theprocedure described by Gorczyca et al. (1992). Briefly, fixed cells werewashed in PBS and resuspended in 50 μl of TdT buffer with 5 units of TdTenzyme (Sigma Chemical Co.) and 0.5 nmol biotin-16-dUTP (BoehringerMannheim Co.). Controls were prepared without TdT enzyme. Cells wereincubated at 37° C. for 1 hour, rinsed in PBS, and resuspended in 100 mlof avidin-FITC, 2.5 mg/ml, (Boehringer Mannheim Co.) in saline-citratebuffer containing 0.1% Triton X-100, 0.1% BSA, 0.5 M NaCl, and 0.06 M Nacitrate. Specimens were incubated in the dark for 30 min, washed in PBSwith 0.1% Triton X-100, resuspended in propidium iodine (5 μg/ml) and0.1% RNAse A. After incubation for 30 min the specimens were analyzedwith the use of an EPICS Profile II flow cytometer (Coulter Corp.,Hialeah, Fla.). An analysis region was set based on the negativecontrols and the percent of labeled cells was calculated from thisregion.

Evaluation of apoptosis. For evaluation of apoptosis induced by theAd-Bax vector, the breast carcinoma cell lines MDA-MB-468, MCF-7, andSKBr3 were used. The cells were plated at 0.5×10⁶ and then treated withAd-Bax or viral control at an MOI of 100 viral particles per cell. Mediaalone was used for mock infection. At 2 and 4 days post transfection,the cells were harvested and fixed in 80% ETOH. After 24 hours,propidium iodide was added to each sample and the cells were analyzed byflow cytometry. The subdiploid cell population was assessed and thepercent recorded as apoptotic cells.

Further analysis of apoptosis was determined by a cell death ELISA kitfrom Boehringer Mannheim. This is a photometric “sandwich enzymeimmunoassay” which allows quantitative in vitro determination ofhistone-associated DNA fragments which are specific for apoptotic celldeath. Briefly, MDA-MB-468 cells and MCF-7 cells were transfected at anMOI of 100 (Ad-Bax, viral control or media alone) and cells collected at72 hours. Samples were incubated with anti-histone biotin and anti-DNAperoxidase in streptavidin coated plates. After removal of unboundantibodies, the amount of peroxidase retained was determinedphotometrically. The results are recorded as an enrichment factor whichis a photometric quantitation above the control samples.

Results

Adp53 Infection Results in Overexpression of p53 Protein and Inductionof p21.

Expression of p53 protein in the H1299 cells was measured at 6 hintervals following Adp53 infection by western blot analysis. Thecontrol cells (mock infected) and dl312 (control vector) infected cellsexpressed no measurable p53 protein. p53 protein was observed at the 6 htime point following infection with Adp53. High expression at multiplephosphorylation states was observed at 24 h and continued to the 36 htime point. Induction of p21 was observed following infection withAdp53. Control cells and dl312 infected cells expressed low levels ofp21 by western blotting analysis. However, induction of p21 was observedearly following infection with Adp53. High levels of p21 were observedat the 18 h time point and continued to with high expression observed at36 h. Similar results were observed at the 24 h time point for the H358and H322J cell lines.

Adp53 Infection Results in a G₁ Cell Cycle Arrest and Induction ofApoptosis.

Cell cycle analysis of the H1299 cell line demonstrated an increase inthe G₁ population of cells following infection with Adp53 compared tothe control and dl312 infected cells (FIG. 2A. This increase in G₁population of cells was observed as early as 12 h following Adp53infection and was clearly evident at the 18 h time point (percent G₁:control=38%, Adp53=59%) and continued to 36 h. Interestingly,accumulation of the sub 2N population of cells was observed at a timepoint slightly delayed from the time of accumulation of cells in G₁ cellcycle arrest. The sub 2N population of cells were observed at 24 hfollowing infection with Adp53 and continued to accumulate up to 36 hfollowing infection. This increase sub 2N population of cellscorresponded to an increase labeling by TUNEL assay (FIG. 2B. These dataare consistent with increases in apoptotic cell death.

Adp53 Infection Result in Decreased Levels of CPP32 and Parp Cleavage.

Levels of the inactive zymogen of CPP32 were observed in control anddl312 infected cells. Adp53 infection resulted in decreased levels ofthe inactive zymogen form of CPP32. These diminished levels of the CPP32zymogen were observed at the 24 h time point and continued through the36 h time point (FIG. 3A). This reduction in CPP32 levels wasaccompanied by concomitant evidence of cleavage of its early target Parpby western blot analysis. Similar results were observed at the 24 h timepoint for the H358 and H322J cell lines (FIG. 3B). The above data isagain consistent with induction of apoptotic cell death, activation ofthe ICE-like protease CPP32, and cleavage of the CPP32 target Parp.

Adp53 Infection Did not Effect the Bcl-2 or Bcl-x_(L) Expression.

No significant changes in the levels Bcl-x, and Bcl-2 proteins wereobserved by western blotting following infection with Adp53 as comparedto control or dl312 infected cells. Similar results were observed at the24 h time point for the H358 and H322J cell lines.

Overexpression of p53 Results in Induction of Proapoptotic Bax and BakProteins.

Bax protein levels were detectable in control and dl312 infected cells.Infection with Adp53 resulted in increased levels of Bax protein. Thiswas especially evident at the 24 h time point and continued to 36 h. Bakprotein expression was detectable by western blot analysis in controland dl312 infected cells. Following infection with Adp53, a significantincrease in Bak protein levels were observed compared to controls.Again, peak levels were present at the 24 h time point and continued tothe 36 h time point. Similar results were observed at the 24 h timepoint for the H358 and H322J cell lines.

Example 2 The Adenoviral Bax Vector

Using the insights gained herein above, the inventors reasoned that theoverexpression of p53 gene induces apoptosis by upregulating Bax. Thusif a vector could be designed that in itself mediated the upregulationof over-expression of Bax, there may be enough of an induction of Bax toinduce apoptosis. In order to investigate this further the inventorsconstructed a new and novel adenoviral Bax vector as described hereinbelow.

Cloning of the Human Bax cDNA.

Total RNA was isolated from SRB I squamous cell carcinoma cell lineusing Ultraspec RNA isolation reagent (Biotecx). First strand cDNA wassynthesized using 5 μg of RNA, 500 ng oligo (dT), 5× strand buffer, 0.1M DTT, 10 μM dNTP mix 1 μl of superscripapt II™ in a RT-PCR™ reaction.Polymerase chain reaction was then performed to amplify Bax cDNA usingforward oligo primer 5′-GGAATTCGCGGTGATGGAC GGGTCCGG-3′ (SEQ ID NO:5)and reverse oligo primer 5′-GGGAATTCTCAGCCCATCTTCTTCCA GA-3′ (SEQ IDNO:6). The reaction was incubated at 95° C. for 1 min, 56° C. for 2 minand 72° C. for 3 min for a total of 35 cycles. The PCR™ reaction wasthen resolved on 1.5% agarose gel. The Bax cDNA sequence was assessedwith the M13 and T7 primers and was found to differ from the wildtypeBax sequence in the amino terminus. The highly conserved BH3 regionwhich appears necessary for apoptosis was intact but a frameshiftmutation existed which eliminated the BH1 and BH2 regions.

Construction of Adenoviral Bax Vector

The TA PCR™II cloning vector (Invitrogen) containing the truncated BaxcDNA (SEQ ID NO:1 cDNA encodes protein of SEQ ID NO:2) was amplified andpurified using Qiagen kit. The truncated Bax gene DNA fragment wasisolated by digestion with restriction enzymes EcoRI (for the 5′ side)and Not I (for the 3′ side) and electroeluted on a 1.5% agarose gel. Thetruncated Bax gene was recovered from the gel with Qiagen DNA recoverykit and inserted into a polylinker between the Xba I and Cla I sites inthe pXCJL.1 shuttle vector. The shuttle vector contains the left end ofthe adenovirus type 5 genome with the E1 region deleted. The resultingplasmid, p12 Bax, was cotransfected with the recombinant plasmid pJM17into 293 kidney carcinoma cells which provided the deleted E1 region inttrqns. pJM17 carries the bulk of the right side of the adenovirus type5 genome.

Calcium phosphate mediated cotransfection of the two plasmids (p12Baxand pJM17) was performed with homologous recombination producing theadenoviral truncated Bax vector (AdBax). Successful adenoviralrecombinants were identified by cytopathic changes in the transfected293 cells. The adenoviral recombinants were amplified on 293 cells andharvested when a complete cytopathic effect was evident. The virus wasisolated by free-thawing the cell pellets three times in dry ice ethanolbath and a 37° C. water bath.

Purification of the virus was performed with two cesium chloridegradient ultracentrifugations. The isolated virus was then dialyzedagainst a buffer (10 mM Tris-HCL, 1 mM MgCl₂ and 10% glycerol) to removecontaminating cesium chloride. Quantification of the virus was thenperformed with O.D. readings ad plaque assay on 293 cells. The purifiedvirus was then analyzed for the presence of the truncated Bax gene bydideoxy DNA sequencing with PCR™ and two primers. The internal forwardoligo primer 5′-GGGACGAACTGGACAGTAA-3′ (SEQ ID NO:7) and reverse oligoprimer 5′-GCACCAGTTTGCTGGCAAA-3′ (SEQ ID NO:8) were used to sequenceboth strands of the adenoviral Bax gene. Additional confirmations wasobtained with PCR™ primers located just upstream and downstream of theBax insert in the adenovirus genome. These primers included the forwardoligo 5′-ACGCAAATGGGCGGTAG-3′ (SEQ ID NO:9) and reverse5′-CAACTAGAAGGCACAGT-3′ (SEQ ID NO:10). Sequencing confirmed that thetruncated Bax gene was correctly inserted in the adenoviral recombinantAdBax.

Example 3 Induction of Apoptosis in Human Breast Cancer by AdenoviralMediated Overexpression of Bax

Apoptosis is controlled, at least in part, by the balance between theproapoptotic (Bax, Bak, Bcl-xs) and antiapoptotic (Bcl-2, Bcl-x_(L))members of the Bcl-2 family. Altering the balance of these mediators canresult in the suppression or induction of apoptosis. The present exampledescribes the use of the novel adenoviral vector, Ad-Bax, to determinewhether overexpression of Bax could induce apoptosis in human breastcancer.

The human Bax cDNA was isolated, sequenced and used to construct theType 5, E1 deleted adenoviral vector as described herein above. TheAd-Bax vector contained a truncated Bax with an intact death (BH3)domain. Human breast cancer cell lines MDA-MB-468, SKBr3 and MCF-7 weretransduced with Ad-Bax, E1 deleted viral control (AdV) or media alone(Cont.) at multiplicity of infection (MOI) of 100 to achieve an 85%transduction efficiency.

Apoptosis was evaluated by changes in cellular morphology, evidence ofDNA-Histone complexes by ELISA and by FACS (FIG. 4A, FIG. 4B and FIG.4C) analysis of subdiploid cells with propidium iodide staining.

Western blot analysis confirmed overexpression of the Bax protein in thetransduced cells. Apoptosis, by morphology, occurred four days aftertransduction with Ad-Bax in 468 and SKBr3 cells but not in MCF-7 cells(Table 5). FACS revealed a two-fold increase in apoptosis (FIG. 4A, FIG.4B, and FIG. 4C). DNA-Histone complexes increased 40% in 468 cells withno increase in MCF-7 cells. Further Western analysis revealed similarlevels of Bcl-xL in all cell lines. However, there were high levels ofBcl-2 only in the apoptosis-resistant MCF-2 cells (Table 5; FIG. 4A,FIG. 4B, and FIG. 4C). TABLE 5 Adenovirally-mediated Bax inducedApoptosis and the Bcl-2 levels in MDA-MB-468, SKBr3 and MCF-7 cell lines% Apoptosis Cell lines Cont AdV. Ad-Bax BCL-2 Level MDA-MB-468 24 26 49Low SKBr3 13 10 24 Low MCF-7 17 12 14 High

The present example demonstrates that adenoviral mediated gene transferof Bax induces apoptosis in human breast cancer cell lines. Resistanceto Ad-Bax induced apoptosis in MCF-7 cells may be due to the highcellular levels of Bcl-2. These results suggest that overexpression ofthe proapoptotic mediator Bax will be a novel and useful gene therapystrategy. Further, such gene therapy may be combined with inhibition ofendogenous Bcl-2 to shift the proapoptotic/antiapoptotic equilibrium infavor of death promotion in cancer cells.

Example 4

Construction of Wild-Type AdBax and AdBak Using a Cosmid System

Traditional methods of producing recombinant adenoviral vectors involveco-transfection of a plasmid encoding the transgene of interest and ashuttle vector carrying adenoviral genome sequences into a cell linesuch as 293 cells that express the E1A gene product. This allows fortransactivation of adenoviral gene transcription and homologousrecombination to produce a recombinant adenovirus that is replicationdeficient. Some drawbacks of this system are a low efficiency ofhomologous recombination, tedious cloning and plaque screening toidentify the desired end product, and the production of a relativelyhigh level of non-recombinant viruses in the viral preparation.

A relatively new method of producing recombinant adenoviral particles isthe use of a cosmid adenoviral vector cloning system (Chartier et al.,1996, Fu and Deisseroth, 1997). The advantages to such a system highrecombination efficiency in recA+ E. coli bacteria, high capacity forheterologous DNA, a stable genome, easy isolation of recombinant virus,and the ability to construct recombinant adenoviruses that carrycytotoxic gene. In the present invention, pro-apoptotic genes such asbax and bak are capable of being introduced into the adenoviral genomeand produced by this system while not killing the producer cell.

The inventors used the Supercos vector (Stratagene, La Jolla, Calif.) asthe base vector for this system (FIG. 5). Initially the SV40 origin ofreplication and the Neo gene were removed by restriction digestion togenerate pCOS/LJ07 (FIG. 6). The cloning of the adenovirus genome in tothe cosmid was attained by cotransfection of pCOS/LJ07 andpAdv-dlE1-dlE3-Gal4 (U.S. application No. 60/030,675, hereinincorporated by reference) into NM522 E. coli cells to allow homologousrecombination to occur. The resultant vector, pCOS/Ad/LJ17 (FIG. 7) waspurified and the recombinant adenovirus then constructed byco-transfection of pCOS/Ad/LJ17 and a shuttle plasmid pCMV/Bak (FIG. 8)into NM522 E. coli cells. The resultant vector pCOS/Ad-Bak (FIG. 9)contains the Bak gene under the control of the CMV IE promoter.Verification of the Ad-CMV-Bak construct by PCR™ confirmed that theproper insert was incorporated into the recombinant virus, andsequencing of the bak gene confirmed the sequence to be wild-type.Similar procedures were used to generate the wild-type bax geneadenovirus recombinant. Linearization of the vector containing theadenoviral genome, and then transfection into 293/GV16 cells results inthe generation of recombinant vectors. FIG. 10, FIG. 11, and FIG. 12outline these procedures.

Thus it is evident that the use of a system such as this is useful forthe construction of adenoviral vectors, and that a wide variety oftransgenes may be incorporated into the adenoviral genome using this orsimilar techniques. It will be appreciated that those of skill in theart may modify or improve such a system to produce better results orachieve greater efficiency.

Example 5 Expression of the Bax Gene by Adenovirus Mediated GeneCo-Transfer

Materials and Methods

Cell lines. Human non-small cell lung cancer cell lines H1299 and A549were cultured in RPMI 1640 and HAM/F12 medium, respectively,supplemented with 10% FBS and antibiotics. Human embryonic kidney 293cells were maintained in Dulbecco's modified Eagle's medium (DMEM)containing 4.5 g/l of glucose with 10% FBS and antibiotics and used inthe construction and amplification of adenovirus vectors.

Construction of recombinant adenovirus vectors. The construction ofAd/PGK-GV16, Ad/GT-Luc, and Ad/GT-LacZ as described by Fang et al.(1998). Ad/CMV-GFP was obtained from Fueyo et al (1998). Mutations foundin the bax cDNA were corrected by combining two PCR products of thegene. The authenticity of the bax-α cDNA sequence was then confirmed byautomatic DNA sequencing performed at M. D. Anderson Cancer Center'sCore DNA Sequencing Facility. For construction of Ad/GT-Bax, the baxgene was first cloned downstream of the GT promoter to generate theshuttle plasmid pAd/GT-Bax. Then, the vector was constructed bycotransfecting 293 cells with a 35-kb cal fragment from Ad/p53 andpAd/GT-Bax (Zhang et al., 1993). The virus titers cited in this studywere determined by optical absorbency at A₂₆₀ (one A₂₆₀ unit=10¹² viralparticle/ml). Particle/plaque ratios usually fell between 30:1 and100:1. All viral preparations were tested for E1⁺ adenoviruscontamination by PCR (Fang et al., 1996) and for endotoxin contaminationby assays with a third-generation pyrogen testing kit from BioWhittaker(Walkersville, Md.).

PCR analysis. Viral DNA was isolated from the supernatant of virusesexpanded in 293 cells. A primer located in the bax gene was then usedwith a second primer located in the adenoviral backbone in PCR toidentify recombinants via PCR. The plasmid pAd/GT-Bax was used as apositive control for Ad/GT-Bax. Primers used for detecting E1⁺adenovirus were the same as in Fang et al. (1996).

Transduction of target cells with adenoviral vectors. All cells wereseeded on 100-mm dishes at a density of 2×10⁶/dish 1 day prior toinfection. H1299 and A549 cells were infected at a total MOI of 900 and1500, respectively. For coadministration of two vectors, the ratio ofthe first vector to second vector was 2:1. A preliminary study showedthat such a ratio resulted in optimal transduction of H1299 cells. Cellswere either harvested at 24 h and 48 h after infection for western blotanalysis or morphological observation by Hoechst staining.

Western blot analysis. Cell samples were lysed or liver samples from thein vivo study were homogenized in a buffer consisting of 62.5 mM Tris,pH 6.8, 6 M urea, 10% glycerol, 2% sodium dodecyl sulfate (SDS), and0.003% bromophenol blue. All samples were sonicated for 30 sec on icebefore the subsequent analysis. Protein concentration was determinedusing BCA Protein Assay Reagent (Pierce, Rockford, Ill.). Fiftymicrograms of protein was mixed with 5% 2-mercaptoethanol, boiled for 5min, and then loaded onto a SDS-polyacrylamide gel. Afterelectrophoresis, the proteins were transferred onto PROTRANnitrocellulose membranes (Schleicher & Schuell, Keene, N.H.), which werethen blocked for 1 h in PBS containing 10% milk. To detect variousproteins, the membranes were probed overnight with primary antibodiesagainst bax (N-20; Santa Cruz Biotechnology, Santa Cruz, Calif.), PARP(C2-10; PharMingen, San Diego, Calif.), caspase-3 (PharMingen), andβ-actin (Amersham, Arlington Heights, Ill.) at concentrationsrecommended by the manufacturers. The membranes were washed 3 times andprobed with horseradish peroxidase-conjugated, species-specificsecondary antibodies (Amersham). Finally, bands were visualized usingthe ECL system (Amersham) according to the manufacturer's instructionsand the density of each band was quantified using Optimas software(Media Cybernetics, Silver Spring, Md.).

Hoechst staining. Cells were seeded on 4-chamber slides at a density of5×10⁴/chamber 1 day prior to infection. Forty-eight hours afterinfection at the MOI described above, cells were fixed with 4%glutaraldehyde and stained with 100 μg/ml Hoechst 33342 (Sigma, St.Louis, Mo.) for 15 min, followed by a gentle washing with PBS.Photographs were taken under a fluorescent microscope.

Animal experiments. Balb/c mice 6-8 weeks old were purchased from theNational Cancer Institute (Frederick, Md.). Prior to injection,Ad/GT-Bax (or Ad/GT-LacZ) was mixed with Ad/PGK-GV16 (or Ad/CMV-GFP) ata ratio of 1:2. A total of 6×10¹⁰ particles/mouse were injected into thetail vein in a volume of 100 μl. Mice were killed 1 day after injection.Their livers were then harvested and frozen at −80° C. for later westernblot analysis or fixed in 10% buffered formalin for later histochemicalanalysis. Sectioning and staining was with hematoxylin and eosin.

Results

Construction of Adenoviruses Expressing Bax.

Shuttle plasmids were constructed in which bax cDNA was driven by GT.Recombinant viral vectors were obtained after a single transfection of293 cells with pAd/GT-Bax plus a 35-kb ClaI fragment from Ad/p53 andidentified by polymerase chain reaction (PCR) analysis with viral DNA.The functionality of Ad/GT-Bax was documented by the coadministration ofAd/GT-Bax and Ad/PGK-GV16 to the cultured human lung carcinoma cell lineH1299 (FIG. 13). Virus from a single plaque was expanded in 293 cellsand twice purified by ultracentrifugation on a cesium chloride gradient.The vector titer determined by optical absorbency at A₂₆₀ was 3.3×10¹²viral particles/ml, equivalent to that of the other E1-deleted vectors,such as Ad/CMV-GFP and Ad/CMV-LacZ. The total yield for Ad/GT-Bax alsowas the same as for the other E1-deleted vectors produced in ourlaboratory, about 1.5×10⁴ particles/cell. The vector preparation wasfree of E1⁺ adenovirus and endotoxin.

Induction of Bax Expression After Adenovirus-Mediated Gene Codelivery.

To demonstrate induction of the bax gene in cultured mammalian cells byadenovirus-mediated gene co-transfer, human lung carcinoma cell linesH1299 and A549 were infected with Ad/GT-Bax and Ad/PGK-GV16 at a vectorratio of 2:1 and at a total multiplicity of infection (MOI) of 900 and1500, respectively. A preliminary experiment showed that this ratio gaveoptimal transduction efficiency in H1299 cells treated at a fixed totalMOI. Cells treated with PBS or infected either with Ad/GT-Bax plusAd/CMV-GFP or with Ad/GT-LacZ plus Ad/PGK-GV16 at the same vector ratioand MOIs were used as controls. Cells were harvested 24 h after thetreatment and their lysates subjected to western blot analysis. Levelsof β-actin in the same western blots were also analyzed and used toensure equal protein loading in all lanes. Though background levels ofthe bax protein expression differed between H1299 and A549 cells andthough the treatment with control vectors did not increase thosebackground levels, a strong induction of bax expression was detected inboth cell lines when they were treated with Ad/GT-Bax plus Ad/PGK-GV16.The induction was seen to be 67.2- and 8.7-fold in H1299 and A549 cells,respectively, when the densities of the bax-specific bands werequantified and normalized to the density of β-actin bands.

Triggering Apoptosis by Induction of the Bax Expression.

Overexpression of the bax gene has been demonstrated to induce therelease of Cyt c from mitochondria (Jurgensmeier et al., 1998; Pastorinoet al., 1998; Rosse et al., 1998) which leads to cleavage first ofcaspase-3/CPP32 followed by cleavage of poly(ADP ribose) polymerase(PARP) (Tewari et al., 1995). To demonstrate the induction of baxexpression and apoptosis by adenovirus-mediated gene codelivery in H1299and A549 cells, samples of the same cell lysate from the above-mentionedexperiments were subjected to western blot analysis of the cleavage ofcaspase-3 and PARP. The cleavage of caspase-3 into a 17-kD fragment andPARP into a 85-kD fragment was detected in cells treated with Ad/GT-Baxplus Ad/PGK-GV16 but not in cells from any other experimental groups. Tofurther document the apoptosis in these cells, H1299 and A549 cells weretreated with various vectors as mentioned above and observed forcytopathology and morphology changes at 48 h after treatment. Over 80%of the cells treated with Ad/GT-Bax plus Ad/PGK-GV16 showed signs ofcytopatholgy, and became rounded and detached, whereas the cells in allother treated groups remained in monolayers with normal morphology.Nuclear fragmentation, a hallmark of cell apoptosis, was detected onlyin cells treated with Ad/GT-Bax plus Ad/PGK-GV16 (FIG. 14), indicatingthat bax expression by this system did activate not only the caspasecascade, but ultimately extensive apoptosis in these human lung cancercell lines.

Induction of Bax Gene Expression In Vivo.

To demonstrate bax gene expression by adenovirus-mediated genecodelivery in vivo, adult Balb/c mice were infused via their tail veinswith PBS, Ad/GT-Bax plus Ad/CMV-GFP, Ad/GT-Bax plus Ad/PGK-GV16, orAd/GT-LacZ plus Ad/PGK-GV16 at a total vector dose of 6×10¹⁰particles/mouse and a vector ratio of 2:1. Mice were then killed at 24 hafter treatment, after which liver samples were harvested for westernblot analysis and histopathological examination. Western blot analysisshowed a 14-fold increase in bax protein levels in animals treated withAd/GT-Bax plus Ad/PGK-GV16, but only background level in all othertreatment groups. These results clearly demonstrated that the Ad/GT-Baxplus Ad/PGK-GV16 strictly regulated bax expression by expressingGAL4/VP16 protein even in vivo. Expression of the bax gene also inducedtypical apoptosis in normal liver cells, as revealed by nuclearfragmentation and condensation in hematoxylin- and eosin-stained liversections (FIG. 15). Together, these results demonstrate thatadenovirus-mediated gene co-transfer can produce sufficient baxexpression and induce apoptosis in vivo.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-38. (canceled)
 39. A method for treating a subject with cancercomprising the steps of: (i) providing an adenoviral expressionconstruct comprising a first nucleic acid encoding a proapoptotic memberof the Bcl-2 gene family and a first promoter functional in eukaryoticcells wherein said nucleic acid is under transcriptional control of saidfirst promoter; and (ii) contacting said expression construct withcancer cells of said subject in a manner that allows the uptake of saidexpression construct by said cells, wherein expression of saidproapoptotic gene results in the treatment of said cancer.
 40. Themethod of claim 39, further comprising contacting said cancer cell witha further cancer therapeutic agent.
 41. The method of claim 40, whereinsaid cancer therapeutic agent is selected from the group consisting oftumor irradiation, chemotherapeutic agent, a second nucleic acidencoding a cancer therapeutic gene.
 42. The method of claim 41, whereinsaid chemotherapeutic agent is a DNA damaging agent selected from thegroup consisting of verapamil, podophyllotoxin, carboplatin,procarbazine, mechlorethamine, cyclophosphamide, camptothecin,ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide(VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin,vinblastin and methotrexate.
 43. The method of claim 41, wherein saidradiation is selected from the group consisting of X-ray radiation,UV-radiation, γ-radiation, or microwave radiation.
 44. The method ofclaim 40, wherein said cancer therapeutic agent comprises a secondnucleic acid.
 45. The method of claim 44, wherein said second nucleicacid is a cDNA or genomic DNA.
 46. The method of claim 44, wherein saidsecond nucleic acid is a second gene operatively linked to a promoter insaid first expression construct.
 47. The method of claim 44, whereinsaid second nucleic acid is a second gene operatively linked to apromoter in a second expression construct.
 48. The method of claim 47,wherein said second expression construct is selected from the groupconsisting of an adenovirus, an adeno-associated virus, a vaccinia virusand a herpes virus.
 49. The method of claim 39, wherein said contactingis effected by regional delivery of the expression construct.
 50. Themethod of claim 39, wherein said contacting is effected by localdelivery of the expression construct.
 51. The method of claim 39,wherein said contacting is effected by direct injection of a tumor withsaid expression construct.
 52. The method of claim 39, wherein saidcontacting comprise delivering said expression construct endoscopically,intratracheally, intralesionally, percutaneously, intravenously,subcutaneously or intratumorally.
 53. The method of claim 39, furthercomprising the step, prior to said contacting, of tumor resection. 54.The method of claim 39, wherein said cancer is selected from the groupconsisting of lung, breast, melanoma, colon, renal, testicular, ovarian,lung, prostate, hepatic, germ cancer, epithelial, prostate, head andneck, pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma,ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph node,small intestine, blood cells, colon, stomach, thyroid, endometrium,prostate, skin, esophagus, bone marrow and blood.
 55. A method ofinhibiting the growth of a cell comprising the steps of: (i) providingan adenoviral expression construct comprising a first nucleic acidencoding a proapoptotic member of the Bcl-2 gene family and promoterfunctional in eukaryotic cells wherein said nucleic acid is undertranscriptional control of said first promoter; and (ii) contacting saidexpression construct with said cell in an amount effective to inhibitthe growth of said cell; wherein expression of said proapoptotic gene bysaid cell results in slower growth of said cell relative to the growthof said cell in the absence of said proapoptotic gene.
 56. The method ofclaim 55, wherein said cell is a cancer cell.
 57. The method of claim56, wherein said inhibition of growth comprises killing of said cancercell.
 58. The method of claim 56, wherein said cancer cell is selectedfrom the group consisting of lung, breast, melanoma, colon, renal,testicular, ovarian, lung, prostate, hepatic, germ cancer, epithelial,prostate, head and neck, pancreatic cancer, glioblastoma, astrocytoma,oligodendroglioma, ependymomas, neurofibrosarcoma, meningia, liver,spleen, lymph node, small intestine, blood cells, colon, stomach,thyroid, endometrium, prostate, skin, esophagus, bone marrow and blood.59. The method of claim 56, wherein said cell is located within amammal.
 60. The method of claim 59, wherein said inhibition of growthcomprises an inhibition of metastatic growth of said cancer cell.
 61. Amethod of inducing apoptosis in a cell comprising the steps of: (i)providing an adenoviral expression construct comprising a first nucleicacid encoding a proapoptotic member of the Bcl-2 gene family andpromoter functional in eukaryotic cells wherein said nucleic acid isunder transcriptional control of said first promoter; and (ii)contacting said expression construct with said cell in an amounteffective to kill said cell; wherein expression of said proapoptoticgene by said cell results in an increase in the rate of death of saidcell relative to the growth of said cell in the absence of saidproapoptotic gene. 62-67. (canceled)
 68. A method for expressing apolypeptide in a target cell comprising introducing into said targetcell: (a) a first vector comprising a coding region for said polypeptideunder the control of a first promoter inducible by an inducerpolypeptide not expressed in said target cell; and (b) a second vectorcomprising a coding region for said inducer polypeptide under thecontrol of a second promoter active in said target cell.
 69. The methodof claim 68, wherein said first and said second vectors are viralvectors.
 70. The method of claim 68, wherein said first and said secondvectors are non-viral vectors.
 71. The method of claim 68, wherein saidfirst vector is a viral vector and said second vector is a non-viralvector, or said first vector is a non-viral vector and said secondvector is a viral vector.
 72. The method of claim 68, wherein saidsecond promoter is a constitutive promoter, an inducible promoter or atissue specific promoter.
 73. The method of claim 69, wherein said viralvectors are the same or different and selected from the group consistingof an adenoviral vector, a herpesviral vector, a retroviral vector, anadeno-associated viral vector, a vaccinia viral vector or a polyomaviral vector.
 74. The method of claim 68, wherein said first vector andsaid second vector are introduced into said target cell at a ratio of1:1, respectively.
 75. The method of claim 68, wherein said first vectorand said second vector are introduced into said target cell at a ratioof 2:1, respectively.
 76. The method of claim 68, wherein said firstvector is introduced at 900 MOI and said second vector at 1500 MOI intosaid target cell.
 77. The method of claim 68, wherein the first promoteris GAL4 and the inducer polypeptide is GAL4/VP16, respectively.
 78. Themethod of claim 68, wherein the target cell is a hyperproliferativecell.
 79. The method of claim 78, wherein said cell is a pre-malignantcell.
 80. The method of claim 78, wherein said cell is a malignant cell.81. The method of claim 80, where said cell is a lung cancer cell, aprostate cancer cell, a brain cancer cell, a liver cancer cell, a breastcancer cell, a skin cancer cell, an ovarian cancer cell, a testicularcancer cell, a stomach cancer cell, a pancreatic cancer cell, a coloncancer cell, an esophageal cancer cell, head and neck cancer cell. 82.The method of claim 68, wherein said first and second vectors areintroduced into said target cell at the same time.
 83. The method ofclaim 68, wherein said first vector is introduced into said target cellprior to said second vector.
 84. The method of claim 83, wherein saidsecond vector is introduced into said target cell within 24 hours ofsaid first vector.
 85. The method of claim 83, wherein said secondvector is introduced into said target cell within 12 hours of said firstvector.
 86. The method of claim 83, wherein said second vector isintroduced into said target cell within 6 hours of said first vector.87. The method of claim 83, wherein said second vector is introducedinto said target cell within 3 hours of said first vector.
 88. Themethod of claim 83, wherein said second vector is introduced into saidtarget cell within 1 hour of said first vector.
 89. The method of claim68, wherein said second vector is introduced into said target cell priorto said first vector.
 90. The method of claim 89, wherein said firstvector is introduced into said target cell within 24 hours of saidsecond vector.
 91. The method of claim 89, wherein said first vector isintroduced into said target cell within 12 hours of said second vector.92. The method of claim 89, wherein said first vector is introduced intosaid target cell within 6 hours of said second vector.
 93. The method ofclaim 89, wherein said first vector is introduced into said target cellwithin 3 hours of said second vector.
 94. The method of claim 89,wherein said first vector is introduced into said target cell within 1hour of said second vector.
 95. The method of claim 78, wherein saidtarget cell is further contacted with a DNA damaging agent.
 96. Themethod of claim 95, wherein said DNA damaging agent is radiotherapy. 97.The method of claim 95, wherein said DNA damaging agent is chemotherapy.98. The method of claim 72, wherein said promoter is an induciblepromoter and the inducing factor is present in said target cell.
 99. Themethod of claim 72, wherein said promoter is an inducible promoter andthe inducing factor is added to said target cell.
 100. The method ofclaim 68, wherein one or both of said vectors further comprise apolyadenylation signal.
 101. The method of claim 68, wherein saidpolypeptide expressed in said target cell is cytotoxic.
 102. The methodof claim 101, wherein said cytotoxic polypeptide is selected from thegroup consisting of an inducer of apoptosis, a cytokine, a toxin, asingle chain antibody, a protease and a antigen.
 103. The method ofclaim 102, wherein said inducer of apoptosis is selected from the groupconsisting of Bax, Bak, Bik, Bim, Bid, Bad and Harakiri.
 104. The methodof claim 103, wherein said inducer of apoptosis is Bax.
 105. The methodof claim 102, wherein said toxin is selected form the group consistingof ricin A-chain, diptheria toxin A-chain, pertussis toxin A subunit, E.coli enterotoxin A subunit, cholera toxin A subunit and pseudomonastoxin c-terminal.
 106. The method of claim 105, wherein said toxin isdiptheria toxin A-chain.
 107. The method of claim 102, wherein saidcytokine is selected form the group consisting of oncostatin M, TGF-β,TNF-α and TNF-β. 108-110. (canceled)
 111. A method of treating a diseasecomprising introducing into cells of a subject having said disease: (a)a first vector comprising a coding region for said therapeuticpolypeptide under the control of a first promoter inducible by aninducer polypeptide not expressed in said target cell; and (b) a secondvector comprising a coding region for said inducer polypeptide under thecontrol of a second promoter active in said target cell.
 112. The methodof claim 111, wherein said disease is selected from the group consistingof lung cancer, prostate cancer, brain cancer, liver cancer, breastcancer, skin cancer, ovarian cancer, testicular cancer, stomach cancer,pancreatic cancer, colon cancer, esophageal cancer and head and neckcancer.
 113. The method of claim 111, wherein said therapeuticpolypeptide is selected from the group consisting of Bax, Bak, Bik, Bim,Bid, Bad, Harakiri, ricin A-chain, diptheria toxin A-chain, pertussistoxin A subunit, E. coli enterotoxin A subunit, cholera toxin A subunit,pseudomonas toxin c-terminal, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.